MEDICAL 


R.F.    TOMLINSON 


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7  fy/fa/wt**^  <st^ 

I  /r 


LABORATORY    MANUAL 


OF 


PHYSIOLOGY 


BY 


FREDERICK  C.  BUSCH,  B.S.,  M.D. 

PROFESSOR     OF    PHYSIOLOGY,     MEDICAL    DEPARTMENT,     UNIVERSITY    OF    BUFFALO 


ILLUSTRATED 


NEW    YORK 

WILLIAM    WOOD    AND    COMPANY 

MDCCCCV 


COPYRIGHT,    1905 
By   WILLIAM    WOOD    AND    COMPANY 


PREFACE. 

IT  has  been  the  aim  of  the  author,  in  compiling  this  little  volume, 
to  give  an  outline  of  experimental  physiology  for  the  guidance  of 
students,  in  a  brief  and  concise  form,  and  sufficiently  comprehen- 
sive in  the  subject-matter  considered. 

Descriptions  of  apparatus  and  illustrations  have,  to  a  large 
extent,  been  omitted,  since  these  are  more  properly  the  function 
of  the  instructor  and  of  the  student  himself.  For  the  same  rea- 
son, results  and  conclusions  of  experiments  have  been  left  to  the 
student  to  work  out  for  himself,  the  descriptions  in  the  text  being 
largely  confined  to  methods  of  procedure  involved  in  obtaining 
the  results. 

The  arrangement  of  chapters  and  experiments  can  be  modified 
to  suit  the  individual  instructor.  The  more  difficult  experiments 
may,  if  desired,  be  given  as  demonstrations.  It  has  been  the  ex- 
perience of  the  author  that  students  work  best  in  groups  of  two 
for  the  simpler  experiments,  and  of  four  for  the  more  complicated 
experiments. 

The  selection  of  the  experiments  presented  has  been  made  with 
a  view  to  the  needs  of  the  medical  student  and  to  the  practical 
application  of  the  first-hand  knowledge,  obtained  in  the  labora- 
tory, to  medical  problems,  later.  At  the  same  time,  it  must  be 
borne  in  mind  that  one  of  the  main  benefits  to  be  obtained  from 
laboratory  work  is  the  training  in  methods  of  exact  observation 
which  the  students  receive. 

The  chapter  on  vision  was  prepared  by  Dr.  Lee  Hasten  Francis, 
to  whom  I  take  this  opportunity  to  express  my  thanks. 

BUFFALO,  N.  Y.,  September,  1905. 


CONTENTS. 

CHAPTER  I. 

BIOLOGICAL  INTRODUCTION. 

PAGE 

Algae i 

Fungi, 4 

Protozoa, 5 

Ciliary  motion, 9 

Effect  of  CO2  on  ciliary  motion, n 

CHAPTER  II. 

MUSCLE-NERVE  PHYSIOLOGY. 

Study  of  electrical  apparatus, 13 

Interrupters, 18 

Dissection  of  frog's  thigh  and  leg, 18 

Elasticity  of  muscle, 20 

Irritability  of  nerve  and  muscle  to  various  stimuli, 22 

Period  of  latent  stimulation  and  form  of  the  single  twitch,      ....  23 

Velocity  of  the  nerve  impulse, 24 

Influence  of  load  on  muscle  twitch, 25 

Influence  of  fatigue  on  the  form  of  the  twitch, 26 

Volume  of  contracting  muscle, 28 

Summation  of  contractions  and  genesis  of  tetanus, 29 

To  determine  actual  shortening  of  contracting  muscle, 31 

Work  done  by  contracting  muscle, 31 

Fatigue  of  human  voluntary  muscle,    .     .     „ 31 

Influence  of  tension  on  muscle  contraction, 32 

Electric  phenomena  of  muscle  and  nerve, 34 

Irritability  and  conductivity  of  nerve  and  muscle  during  and  after  the 

passage  of  a  constant  current  (electrotonus), 38 

The  constant  current  as  a  stimulus  (Pfliiger's  laws), 41 

Stimulation  of  human  nerves, 42 

Reaction  of  degeneration, 44 

[v] 


CONTENTS. 

PAGE 

Action  of  drugs  on  the  muscle  twitch,      . ' 45 

Involuntary  muscle, 45 

CHAPTER  III. 

NERVOUS  SYSTEM. 

Reflex  action, 47 

Reaction  time, 50 

Removal  of  the  cerebrum  of  the  frog, 51 

Removal  of  the  cerebrum  of  the  pigeon, 52 

Removal  of  the  cerebellum  of  the  pigeon, 52 

Hemisection  of  the  spinal  cord, 52 

Stimulation  of  the  motor  area  of  the  dog's  brain, 53 

Division  of  the  semicircular  canals, 54 

Determination  of  the  number  of  impulses  discharged  by  a  nerve  ceil  in 

a  unit  of  time,     . 55 

CHAPTER  IV. 

BLOOD. 

Coagulation  of  the  blood, 56 

The  number  of  red  and  white  corpuscles, 59 

Changes  produced  in  the  corpuscles  by  variation  of  osmotic  pressure,  62 

Microscopic  examination, 63 

Staining  the  blood  cells, 64 

Classification  of  the  leucocytes, 66 

Differential  count  of  the  leucocytes, 66 

Estimation  of  the  haemoglobin, 68 

Estimation  of  the  specific  gravity, 71 

Haemoglobin  and  its  derivatives, 73 

Spectra  of  haemoglobin  and  its  compounds, 75 

Globulicidal  action  of  serum, 77 

CHAPTER  V. 

CIRCULATION  or  THE  BLOOD. 

Study  of  the  circulation  in  the  frog's  web, 80 

Migration  of  the  leucocytes, 81 

Direct  observation  of  the  action  of  the  frog's  heart, 82 

Graphic  record  of  the  frog's  heart-beat, 84 

Influence  of  temperature  upon  the  beat  of  the  frog's  heart,    ....  85 

[vi] 


CONTENTS. 

PAGE 

Dissection  of  the  extrinsic  cardiac  nerves  of  the  frog, 86 

Effect  of  stimulation  of  the  vago-sympathetic  trunk  upon  the  heart-beat,  87 

Reflex  inhibition  of  the  frog's  heart, 88 

Effect  of  drugs  upon  the  heart's  action, 88 

Perfusion  of  frog's  heart, 92 

Action  of  certain  salts  upon  the  heart  muscle, ...  94 

Stannius'  experiment, 95 

Maximal  response  of  heart  muscle  to  minimal  stimulus, 96 

Refractory  period, 97 

Dissection  of  mammalian  heart, 97 

Dissection  and  relations  of  the  extrinsic  cardiac  nerves, 98 

Direct  observation  of  the  pulsating  mammalian  heart, 98 

Stimulation  of  extrinsic  cardiac  nerves, 100 

Action  of  the  heart  valves, 101 

Mechanics  of  the  circulation, 102 

Pulse  record,  in  man, 106 

Volume  pulse, 107 

Apex  beat,  cardiogram,  and  heart  sounds, 109 

The  vasomotor  mechanism, no 

Record  of  the  blood  pressure  in  the  rabbit, 113 

Perfusion  of  the  rabbit's  heart, 117 

Effect  of  temperature  on  the  heart-beat, 118 

Estimation  of  human  blood  pressure, .  118 

CHAPTER  VI. 
SECRETION.     DIGESTION.     ABSORPTION. 

Secretion  of  saliva, 121 

Changes  in  the  gland  cells  following  chorda  stimulation,    .     .     .     .     .124 

Salivary  digestion, " 125 

Mechanism  of  swallowing, 127 

The  vagus  as  a  motor  nerve  to  the  stomach, 129 

Gastric  digestion, 130 

Intestinal  digestion, 134 

Mechanism  of  pancreatic  secretion, 138 

CHAPTER  VII. 

INTERNAL  SECRETIONS. 

Liver,  glycogen, 140 

Pancreatic  diabetes, 141 

[vii] 


CONTENTS. 

PAGE 

Thyroid,  removal  of, 142 

Thyroid  feeding  after  thyroid  removal, 144 

Suprarenal  glands, 144 

Ablation  of  the  suprarenal  in  the  rabbit, 144 

Suprarenal  extract,  action  of, 145 

CHAPTER  VIII. 

RESPIRATION. 

Respiratory  movements, 148 

Respiratory  sounds.     Auscultation, 149 

Palpation.     Vocal  fremitus, 150 

Percussion, 150 

Chest  measurements, 151 

Respiratory  capacity, 151 

Cardio-pneumatic  movements, 152 

Pulmonary  pressure, 153 

Intrathoracic  pressure,        153 

Vagus  nerve  in  respiration, .  154 

Innervation  of  the  diaphragm, 156 

Effect  of  blood  temperature  on  respiration,       158 

Effect  of  anaemia  upon  the  respiratory  centre, 159 

Respiratory  centre, 160 

Condition  of  lung  following  section  of  both  vagi, 161 

Artificial  respiration, 162 

Estimation  of  COa  and  H2O  in  expired  air, 163 

CHAPTER  IX. 
EXCRETION. 

Movements  of  the  ureter  and  bladder, 166 

Urine  flow.     Kidney  volume, 167 

Blood  pressure  and  kidney  volume, 168 

Intravenous  injection  of  dextrose, 169 

Intravenous  injection  of  albumin, 169 

Intravenous  injection  of  peptone, 170 

Effect  of  peptone  upon  the  coagulability  of  the  blood, 170 

CHAPTER  X. 

SENSATION. 

General  consideration, 171 

Quantitative  relation  between  stimulus  and  sensation, 172 


CONTENTS. 

PAGE 

Weber's  law, 173 

Cutaneous  sensation,      .     .     .     , 173 

Temperature  sense, 176 

CHAPTER  XI. 
VISION. 

Dissection  of  the  eye, 178 

Physiological  optics, , 179 

Miscellaneous  experiments,     . 187 

Normal  vision, 188 

Abnormal  vision, 190 

Correction  of  refractive  defects, 191 

Ophthalmoscopy, 192 

Perimetry, 193 

Drugs  acting  locally  on  the  eye, 194 


[ix] 


LABORATORY  MANUAL  OF  PHYSIOLOGY. 

CHAPTER  I. 

BIOLOGICAL  INTRODUCTION. 

Appliances. — An  aquarium;  Slides;  Cover  glasses;  Test-tubes;  Pipettes; 
Beakers;  Microscope. 

SINCE  the  basis  of  physiology  as  well  as  morphology  is  the  cell, 
a  few  examples  of  the  more  common  simple  plant  and  animal  cells 
are  here  presented  for  study,  as  a  preparation  to  the  observation 
of  the  physiologic  phenomena  accompanying  the  activity  of  the 
more  highly  differentiated  cell-groups  of  the  higher  animals.  • 

For  this  purpose,  well-known  representatives  of  the  algae,  fungi, 
and  protozoa  have  been  chosen. 

I.  ALG.E. 

These  are  plant  cells  of  the  lowest  order,  consisting,  either  of 
single  cells  leading  an  individual  existence,  or  of  groups  of  cells 
attached  end  to  end  so  as  to  form  filaments  or  threads.  A  green 
coloring  matter,  chlorophyll,  is  common  to  the  group. 

1.  Protococcus. — In  the  mud  of  shallow  pools,  ditches,  and 
roof-gutters,  a  unicellular  form,  Protococcus,  may  commonly  be 
found.  This  is  seen,  in  the  vegetative  stage,  as  a  spheroidal  body,  of 
small  size,  having  an  outer  tough  transparent  envelope,  composed, 
chiefly,  of  cellulose,  and  enclosing  viscid  granular  protoplasm. 
Certain  portions  of  the  protoplasm  contain  the  coloring  matter, 
chlorophyll,  which  may  be  either  green  or  red.  These  portions  are 
known  as  chromatophores.  The  cell  contains  a  distinct  nucleus 
with  a  nucleolus.  Reproduction  takes  place  by  the  formation  of 
so-called  zoospores.  These  are  of  two  kinds,  macro-  and  micro- 


LABORATORY  MANUAL  OF  PHYSIOLOGY. 

spores.  The  former  are  produced  by  the  division  of  the  cell  con- 
tents into  two  or  four  ovoid  masses.  These  are  set  free  through 
the  resorption  of  the  mother  cell  membrane,  develop  two  cilia  at 
opposite  poles,  and  become  free  swimming  forms.  Later,  a  cell 
envelope  is  also  formed.  The  microspores  are  smaller,  more  nu- 
merous, and  are  devoid  of  cell  wall.  Both  forms,  finally  come  to 
rest,  lose  their  cilia,  develop  a  thick  cell  wall,  and  again  assume 
the  vegetative  condition. 

Sunlight  is  essential  to  the  growth  of  this  plant  as  it  is,  in  fact, 
to  all  chlorophyll  plants.  It  is  through  the  agency  of  the  chloro- 
phyll that  carbon  dioxide  is  broken  down  into  carbon  and  oxygen 
for  the  constructive  metabolism  of  the  plant.  This  is  a  distinctive 
characteristic  of  the  algae  and  other  chlorophyll  plants  as  com- 
pared with  the  non-chlorophyll  plants,  such  as  the  fungi. 

Practicum. — Spread  out  some  mud,  containing  protococcus,  on 
a  glass  slide,  dilute  with  water,  and  look  for  the  plant  with  a  low 
power  of  the  microscope.  Study  with  a  high  dry  lens.  Make 
out  the  following  points-  size;  form;  structure;  zoospores.  Stain 
with  iodine.  This  kills  the  cell  and  may  show  the  cilia. 

Into  two  tubes,  filled  with  and  inverted  over  mercury,  introduce 
some  water  rich  in  protococcus.  From  a  carbon  dioxide  genera- 
tor, introduce  into  each  tube  a  few  bubbles  of  the  gas.  Place  one 
tube  in  the  dark  and  the  other  in  the  light.  After  several  hours, 
examine  the  tubes.  Measure  the  gas  in  both.  Place  a  small  piece 
of  KOH  in  each  tube.  Is  the  gas  absorbed  ?  In  the  tube  that  has 
been  in  the  light,  introduce  a  few  drops  of  pyrogallic  acid  solution. 
Is  any  more  gas  absorbed  ?  Explain. 

Place  some  water,  containing  numbers  of  zoospores,  on  a  slide; 
cover  with  a  long  cover  slip  and  place  under  the  microscope.  With 
the  substage  mirror,  cause  a  beam  of  bright  sunlight  to  pass 
through  the  specimen.  What  is  the  effect  on  the  movement  of  the 
spores  ?  Now  reduce  the  intensity  of  the  light.  What  is  the  effect  ? 

2.  Spirogyra. — This  form  is  commonly  found,  during  the  sum- 
mer, in  ponds  and  tanks,  as  floating  masses  of  a  light  green  color. 
These  masses  are  found,  on  observation,  to  consist  of  long,  fine 


BIOLOGICAL  INTRODUCTION. 

green  threads.  Each  thread  is  made  up  of  a  number  of  cylindrical 
cells  placed  end  to  end.  In  each  cell  there  are  one  or  more  spiral 
bands  of  a  bright  green  color.  These  contain  the  chlorophyll  of 
the  plant  and  are  called  chromatophores.  At  intervals  in  the  band, 
small,  round  bodies  may  be  seen  which  contain  proteid  substance. 
Aside  from  the  spiral  bands  and  the  thin  layer  of  protoplasm  lin- 
ing the  cellulose  cell  wall,  the  cell  space  is  filled  with  so-called  cell 
sap.  This  consists  of  water  in  which  certain  inorganic  and  organic 
substances  are  dissolved.  The  nucleus  may  be  either  central  or 
peripheral.  The  growth  of  the  plant  is  accomplished  by  cell 
division  in  the  long  axis,  maintaining  the  thread  formation. 

Reproduction  takes  place  through  conjugation.  This  is  a  good 
example  of  sexual  reproduction.  Ordinarily  this  occurs  in  the  fol- 
lowing manner.  Cells  from  two  adjoining  filaments  send  out  pro- 
trusions toward  each  other.  These  meet  and  join,  their  adjoining 
membranes  becoming  absorbed  so  as  to  form  one  continuous  tube. 
The  cell  contents  of  both  contract,  the  one  before  the  other,  and 
the  contents  of  one  cell  run  through  the  tube  into  the  other,  nucleus 
uniting  with  nucleus,  chromatophore  with  chromatophore.  This 
is  typical  of  sexual  reproduction  in  both  plants  and  animals.  In 
this  case,  the  new  cell  thus  formed  is  called  a  zygospore.  This, 
which  is  at  first  spherical  in  shape,  without  any  distinct  cell  wall, 
increases  in  size  by  the  imbibition  of  water,  assumes  the  form  of  an 
ellipse  and  develops  a  hard  envelope  which  is  impermeable  to 
water.  In  this  condition  the  cell  can  withstand  drying  and  con- 
siderable variations  in  temperature.  When  the  plant  again  ger- 
minates, the  cell  wall  is  burst  and  the  contents  grow  out  into  a  new 
filament. 

Practicum. — Observe,  with  unaided  eye,  a  mass  of  spirogyra. 
Mount  a  few  filaments  and  observe  under  first  low  and  then  high 
power  of  the  microscope.  Describe  the  structure.  Make  a  draw- 
ing. Stain  a  specimen  with  carmine,  after  fixation  in  picric  acid. 

To  observe  the  phenomena  of  reproduction,  examine  a  tresh 
specimen  that  has  been  kept  in  the  cold,  over  night. 

Observe,  from  time  to  time,  a  specimen  that  has  been  kept  in 

[3] 


LABORATORY  MANUAL  OF  PHYSIOLOGY. 

distilled  water.    What  is  the  effect  on  the  life  and  growth  of  the 
plant  ? 

Place  a  specimen  in  the  dark  for  several  hours.  Examine  for 
starch  by  treating  with  iodine.  Result  ?  Place  some  of  this  same 
material  in  bright  sunlight  for  several  minutes.  Examine  for 
starch  again.  Result  ? 

II.  FUNGI. 

Yeast. — (Torula  or  Saccharomyces  cerevisice). — This  plant,  in 
common  with  the  other  fungi,  differs  from  the  algae,  which  have 
been  studied,  in  not  containing  any  chlorophyll.  The  algae,  which 
contain  this  pigment,  are  able  to  obtain  their  nutrition  from  the  in- 
organic constituents  of  their  environment.  The  yeast,  on  the  other 
hand,  in  which  this  pigment  is  absent,  is  dependent  upon  organic 
material  for  the  processes  of  its  metabolism. 

Practicum. — Nutritive  fluid  for  yeast  (Pasteur's  fluid). 

Potassium  phosphate 2.0  grams. 

Calcium  phosphate 0.2        " 

Magnesium  sulphate 0.2       " 

Ammonium  tartrate 10.0        " 

Cane  sugar 150-0       " 

Water to  1000.0       " 

(a)  Put  a  small  quantity  of  fresh  baker's  yeast  into  some  of  the 
above-described  fluid  and  keep  in  a  warm  place.    As  soon  as  the 
culture  becomes  frothy  and  cloudy  it  is  ready  for  examination. 

(b)  Place  some  of  this  mixture  on  a  slide  without  a  cover  glass 
and  examine  with  a  low  power  of  the  microscope.    Note  size  and 
arrangement  of  the  cells.     Place  a  cover  slip  on  the  specimen  and 
examine  with  a  high  power.    What  is  the  mode  of  union  of  the 
cells  ?    Describe  the  structure.    Make  a  drawing. 

(c)  Stain  a  specimen  with  fuchsin.     Treat  another  with  iodine. 
Is  there  any  starch  present  ? 

(d)  Sow  some  yeast  in  Pasteur's  fluid  and  place  in  the  incuba- 
tor for  several  hours.    Place  another  specimen  in  the  cold  for  the 
same  length  of  time.    Compare  the  growth  in  the  two  specimens. 

[4] 


BIOLOGICAL  INTRODUCTION. 

(e)  Take  two  other  specimens,  keeping  one  in  the  dark,  the 
other  in  the  light.  Is  there  any  change  in  growth  ? 

(/)  In  a  sterilized  test-tube,  place  some  yeast  mixture  and  boil 
for  several  minutes.  Replace  the  sterile  cotton  plug  in  the  mouth 
of  the  tube  and  set  to  one  side.  Examine  from  time  to  time.  Does 
growth  continue  ? 

(g)  Taste  some  fresh  yeast  mixture.  Taste  again  after  the 
mixture  has  been  in  the  incubator  for  twenty-four  hours.  To 
what  is  the  difference  in  taste  due? 

(h)  Grow  some  yeast  in  a  tightly  stoppered  flask,  connected,  by 
means  of.  a  bent  glass  tube,  with  another  flask  containing  a  solu- 
tion of  calcium  hydrate.  What  is  the  result  ?  Explain. 

III.  PROTOZOA. 

1.  Amoeba. — These  simplest  forms  of  animal  life  are  distin- 
guished from  the  simplest  plant  cells,  not  so  much  through  their 
power  of  locomotion  as  through  a  difference  in  their  processes  of 
nutrition.  The  plant  cells,  which  have  been  studied  in  the  previ- 
ous exercises,  have  been  able  to  build  up  their  living  cell  substance, 
protoplasm,  a  proteid  body,  out  of  non-proteid  material.  This  the 
animal  cells,  of  which  the  amceba  is  a  type,  are  unable  to  do.  In 
other  words,  the  animal  cells  are  dependent  upon  vegetable  cells 
to  manufacture  their  protein  for  them. 


•  4 


FIG.  i.— Amceba.    Phases  of  amoeboid  movement. 


Amceba  are  commonly  found  in  stagnant  pools,  mud,  and  col- 
lections of  water  containing  decaying  vegetable  matter.  They  are 
seen  as  minute  jelly  like  masses,  with  a  more  or  less  granular  in- 
terior and  a  clearer,  more  transparent  peripheral  portion  (see 
Fig.  i). 

[5] 


LABORATORY  MANUAL  OF  PHYSIOLOGY. 

Under  certain  conditions,  they  assume  a  spherical  form.  Gen- 
erally, however,  they  are  seen  undergoing  constant  changes  of 
form  which,  at  times,  may  be  very  rapid.  These  changes  consist  in 
the  protrusion,  from  various  parts  of  the  outer  portion  or  ectosarc 
of  the  animal,  of  processes  into  which  the  more  fluid  interior  por- 
tion flows.  Such  processes  are  called  pseudopodia.  By  the  forma- 
tion of  pseudopodia,  the  animal  is  enabled  to  move  about.  This, 
then,  is  a  form  of  locomotion. 

The  amoeba  may  contain  one  or  more  nuclei,  usually  only  one. 
In  some  part  of  the  outer  portion,  a  large,  clear  space  in  the  proto- 
plasm is  seen,  alternately,  to  grow  larger  and  smaller.  This  is  the 
contractile  vesicle  or  vacuole. 

Practicum. — (a)  Place  a  drop  of  amoebae-containing  water,  on  a 
slide.  Cover  with  a  supported  cover  slip.  Examine  with  a  low 
power.  Having  found  an  amceba,  examine  it  with  the  high  power. 
Make  a  drawing,  indicating  its  main  points  of  structure.  Watch 
an  active  specimen  and  make  outline  drawings  from  time  to  time 
to  show  the  change  in  form.  Look  for  a  specimen  that  is  ingesting 
food.  What  is  the  process  ? 

(b)  Observe  the  effect  of  heat  upon  the  movements  by  using  the 
hot  stage. 

(c)  In  the  same  way  that  you  have  studied  the  amceba,  study 
the  white  blood  corpuscles  of  the  frog  and  of  man.    To  bring  out 
the  nuclei,  treat  with  dilute  acetic  acid.    The  slides  for  the  study 
of  the  living  cells  of  man  must  be  kept  on  a  warm  stage. 

(d)  Into  the  dorsal  lymph  sac  of  a  frog,  inject  an  aqueous  mixt- 
ure of  carmine  or  a  fine  suspension  of  lamp-black.    After  fifteen 
minutes  or  a  half  hour,  withdraw  some  lymph  and  examine  for  the 
ingestion  of  foreign  bodies  by  the  white  cells. 

2.  Infusoria. — These  are  protozoa  having  bodies  of  a  constant 
form  and  depending,  for  locomotion,  upon  flagella  or  cilia.  The 
flagella  may  be  single  or  double  and  may  be  attached  to  one  or 
both  poles  of  the  organism.  One  of  the  most  common  found  in 
aquaria  is  Euglena  viridis,  having  a  long  whip-like  flagellum  at  one 
end,  by  means  of  which  it  swims  rapidly  through  the  water. 

[6] 


BIOLOGICAL  INTRODUCTION. 

The  lowest  forms  of  flagellata  are  the  trypanosomata,  a  parasitic 
type  of  which  is  shown  in  Fig.  2.  These  forms  have  lately  be- 
come of  medical  interest  because  of  their  apparent  causal  relation 
to  certain  tropical  diseases. 

Common  examples  of  the  ciliated  protozoa  that  are  found  in 


FIG.  2.— Frog's  Blood.    A,  Trypanosome ;  B,  eosinophile.    (Williams.) 

ponds  and  stagnant  pools  are  Paramecium  and  .Vorticella  (see 
Figs.  3  and  4). 

The  former  is  a  free  swimming  form,  rather  oval  and  somewhat 
flattened  in  shape,  with  a  mouth-like  aperture  at  one  side,  leading 
into  a  short  stomach-like  pouch.  The  animal  is  covered  with  cilia 
which  are  longer  in  the  mouth  region  and  at  the  posterior  end. 
A  nucleus  can  be  distinguished,  as  well  as  two  contracting  vesicles, 
one  at  either  end  of  the  animal. 

The  Vorticellae  are  seen  as  solitary  individuals,  consisting  of  an 
oval  body  mounted  on  a  long  stalk  which  is  attached  to  some  for- 
eign body.  At  the  free  end,  a  flattened  disc  surrounded  by  cilia  is 
seen.  At  one  side  of  this  is  the  aperture  known  as  the  mouth. 

On  closer  examination,  it  is  seen  that  the  band  of  cilia  does  not 


LABORATORY  MANUAL  OF  PHYSIOLOGY. 


form  a  complete  circle,  but  rather  a  spiral,  which  passes  down  the 
tube  leading  from  the  mouth  and  known  as  the  esophagus.  There 
is  a  sausage-shaped  or  spiral  nucleus  and  a  contracting  vacuole. 
The  stalk  may  be  seen  under  the  high  power  to  contain  a  core  of 
contractile  protoplasm. 

Practicum. — (a)  Place  a  little  powdered  carmine  in  the  water 
containing  vorticellae.    Examine  under  the  microscope.    What  be- 


FlG.  3. — Paramecium.  i 
and  2,  Contracting  vacu- 
oles ;  3,  nucleus  ;  4,  mouth  ; 
5,  stomach  ;  6,  food  parti- 
cles. 


A. 

FIG.  4— Vorticella.  A,  Open,  stalk  extended 
B,  closed,  stalk  coiled,  n,  Nucleus ;  v,  vacuole 
f,  food  particles. 


comes  of  the  carmine  ?  Note  the  motion  of  the  cilia.  Follow  the 
movement  of  the  food  vacuoles.  Note  the  movements  of  the  ani- 
mal as  a  whole.  What  is  the  function  of  the  stalk  ?  If  opportunity 
offers,  observe  the  process  of  multiplication. 

(b)  Effect  of  various  gases  upon  the  activity  of  protozoa.     Car- 
bon dioxide.    Arrange  a  deep-celled  slide  in  such  a  way  that  a 

[8] 


BIOLOGICAL  INTRODUCTION. 

stream  of  gas  from  a  CO2  generator  may  be  made  to  pass  through. 
Place  a  small  drop  of  water  from  the  aquarium  containing  proto- 
zoa, upon  a  cover  slip.  Cover  the  top  of  the  cell  on  the  slide  with 
a  thin  layer  of  vaseline.  Invert  the  cover  slip  over  the  cell.  The 
vaseline  serves  to  cement  the  slip  to  the  cell  and  makes  an  air- 
tight compartment.  Place  this  hanging  drop  slide  upon  the  stage 
of  the  microscope.  Study  with  both  low  and  high  power.  Make 
out  the  different  organisms  present.  Carefully  observe  the  condi- 
tion of  activity  of  the  organisms  present.  Now  connect  up  the  CO2 
generator  and  allow  a  stream  of  the  gas  to  pass  through  the  hang- 
ing-drop cell.  What  is  the  effect  upon  the  activity  of  the  protozoa  ? 
Compare  this  with  the  relation  of  the  carbon  dioxide  to  plant  me- 
tabolism. Stop  the  passage  of  the  gas  through  the  cell,  draw  fresh 
air  through  and  observe  again. 

(c)  With  a  new  hanging  drop  and  slide,  try  the  effect  of  the  va- 
pors from  various  volatile  substances,  such  as  chloroform,  ether, 
alcohol,  ammonia,  etc.  This  may  be  accomplished  by  placing  a 
small  pledget  of  cotton,  saturated  with  the  substance,  in  the  bot- 
tom of  the  cell.  To  see  if  the  effect  of  these  gases  is  permanent  or 
not,  remove  the  cover  slip  from  the  slide  and  mount  on  a  fresh 
slide. 

IV.  CILIARY  MOTION  AS  SEEN  IN  CILIATED  EPITHELIUM. 

One  of  the  most  convenient  objects  for  this  purpose  is  the  man- 
tle of  the  oyster. 

1 .  Remove  a  small  portion  of  the  edge  of  the  mantle  of  an  oyster, 
mount  on  a  slide,  and  study  with  high  and  low  power  of  the  micro- 
scope.   Is  the  ciliary  motion  equally  extensive  in  both  directions  ? 
Is  it  equally  rapid  in  both  directions  ? 

2.  The  rate  of  ciliary  movement  may  be  ascertained  by  means 
of  the  following  device.     Arrange,  under  the  stage  of  the  micro- 
scope, a  time-marker  with  a  paper  flag  attached  to  its  writing  lever. 
This  is  so  adjusted  that  the  movements  of  the  flag  rhythmically 
interrupt  the  passage  of  the  light  through  the  specimen  under  ex- 
amination.   By  the  use  of  a  metronome,  vibrating  rods,  or  tuning 

[9] 


LABORATORY  MANUAL  OF  PHYSIOLOGY. 

forks,  the  time-marking  lever  may  be  caused  to  vibrate  any  desired 
number  of  times  per  second. 

The  number  of  interruptions  is  varied  until  a  frequency  is  ob- 
tained which  shuts  off  the  light  in  such  a  manner  that  the  cilia 
appear  stationary.  This  frequency  corresponds  to  the  rate  of  vi- 
bration of  the  cilia. 

3.  Pith  a  Frog. — This,  ordinarily,  means  destroying  the  brain, 
and  is  accomplished  in  the  following  manner:  Hold  the  frog  in  the 
left  hand,  securing  the  head  between  the  index  and  middle  fingers, 
with  the  thumb  over  the  back.  Bend  the  head  forward  so  as  to 
place  the  occipito-atlantal  ligament  on  the  stretch  and  expose  the 
articulation  between*  the  skull  and  the  vertebral  column.  With 
fine  scalpel  or  fine  pointed  scissors,  make  an  incision  through  the 
neuraxis  at  this  point.  Run  a  blunt-pointed  seeker  through  this 
opening  into  the  cranial  cavity  and  destroy  the  brain.  If  required, 
the  cord  may  be  broken  up  in  a  similar  manner. 

(a)  With  heavy  scissors,  cut  off  the  lower  jaw  of  the  frog.    Place 
the  frog  on  its  back ;  wash  off  the  mucous  membrane  of  the  roof  of 
the  mouth  with  normal  salt  solution ;  remove  the  excess  with  filter 
paper;  place  a  small  piece  of  cork  on  the  mucous  membrane  near 
the  apex  of  the  jaw  and  watch  its  movement.    Time  the  movement 
of  the  cork  for  a  certain  distance. 

(b)  Repeat  the  experiment  after  having  bathed   the  mucous 
membrane  with  warm  salt  solution  (36°  C.). 

(c)  Repeat  again  after  bathing  with  cold  salt  solution  (5-10°  C.). 
What  is  the  effect  of  temperature  upon  ciliary  motion  ? 

(d)  The  above  experiment  may  be  repeated,  using  the  appa- 
ratus shown  in  Fig.  5.    For  this  purpose,  dissect  out  a  piece  of 
the  frog's  oesophagus;  pin  it,  outer  side  down,  upon  a  cork  board; 
adjust  the  weight  (W)  and  the  lever  (P)  so  that  the  rate  of  ciliary 
movement  may  be  indicated  on  the  graduated  arc  (S). 

(e)  Using  the  same  apparatus,  try  the  effect  of  various  weights 
upon  the  ciliary  motion,  beginning  with  five  grams  and  increasing 
the  weight  until  the  cilia  are  no  longer  able  to  move  it. 

(/)  Repeat  this  experiment  with  a  fresh  preparation,  tilting  the 

[W] 


BIOLOGICAL  INTRODUCTION. 

cork  board  at  an  angle  of  about  thirty-five  degrees.  Flat  weights 
will  be  necessary  on  account  of  the  incline.  Measure  the  height  of 
the  upper  edge  of  the  preparation  from  the  lower  edge.  Observe 
the  time  taken  for  the  passage  of  a  given  weight  through  this  dis- 


FIG.  5.— To  Show  Ciliary  Motion.    (According  to  Kronecker.)    E,  Portion  of  frog's 
oesophagus  ;  W,  cork  with  weight ;  P,  pointer ;  S,  scale. 


tance.  Estimate  the  work  by  the  cilia,  according  to  the  following 
formula:  W,  work  done;  G,  weight  in  milligrams;  H,  height  in 
millimetres.  W,  then,  would  equal  G  multiplied  by  H.  What  is 
the  work  done  per  second  ?  Per  minuute  ? 

4.  Effect  of  C02  on  Ciliary  Motion. — (a)  Fill  a  small  bell  jar 
with  water  and  invert  over  water.  Introduce  a  tube  from  a  CO2 
generator  and  fill  the  jar  with  the  gas.  Pin  an  oesophagus  prepara- 
tion to  a  cork  board  and  place  on  a  glass  plate  greased  with  vase- 
line. Place  a  cork  with  weight  upon  the  preparation.  Quickly 
adjust  the  gas  containing  jar  over  the  ciliary  preparation.  Ob- 
serve the  rate  of  ciliary  motion,  as  before.  What  is  the  effect  of  the 
gas? 


LABORATORY  MANUAL  OF  PHYSIOLOGY. 

(b)  Repeat  the  above  experiment,  placing  a  piece  of  absorbent 
cotton,  saturated  with  chloroform,  under  the  jar.    Effect  ? 

(c)  Repeat,  using  ether.    Ammonia.    Other  gases. 

5.  Keep  a  piece  of  frog's  oesophagus,  moistened  with  normal 
saline,  for  several  days.  From  time  to  time  scrape  off  a  bit  of  the 
epithelium  and  examine  under  the  microscope  for  active  cilia. 

How  long  do  the  cilia  survive  after  the  death  of  the  frog  ?  What 
is  the  function  of  ciliated  epithelium  ?  Where  is  it  found  in  man  ? 


[12] 


CHAPTER  II. 

MUSCLE-NERVE. 

Appliances. — Revolving  drum;  Myograph  (heavy);  Light  muscle  lever; 
Inductorium;  Moist  chamber;  Platinum  electrodes;  Non-polarizable  elec- 
trodes; Rheostat;  Rheocord;  Rheonome;  DuBois  keys  (2);  Pohl's  commu- 
tator; Glass  plate  for  holding  nerve;  Cut-out  key;  Capillary  electrometer; 
Current  interrupters;  Signal  magnets;  4  dry  cells;  Light  copper  wire;  Va- 
rious chemical  reagents  (see  text  below). 

I.  STUDY  OF  ELECTRICAL  APPARATUS. 

1.  The  Galvanic  Current. — Xhe  fundamental  experiment  upon 
which  the  principles  of  this  form  of  electricity  have  been  based  was 
unwittingly  performed  by  Galvani  in  1786.  Galvani  noted  that 
some  frog's  shanks,  which  had  been  hung  by  copper  wires  from  an 
iron  railing  and  which  were  swinging  to  and  fro,  twitched  when- 
ever they  came  in  contact  with  the  railing.  It  is  true  that  Galvani 
misinterpreted  the  results  of  this  particular  experiment,  ascribing 
the  phenomena  to  the  development  of  an  electric  current  within  the 
tissues  themselves.  That  there  are  tissue  currents,  he  did  show  by 
later  experiments. 

It  was  demonstrated,  however,  by  Volta,  a  contemporary,  that 
Galvani's  initial  experiment  was  due  to  the  production  of  a  cur- 
rent through  completing  the  circuit  between  two  metals  of  a  differ- 
ent potential.  This  constant  flow  of  current  between  two  sub- 
stances of  different  potential  has  been  called  galvanic  electricity, 
in  contradistinction  to  that  form  consisting  of  a  single  discharge  or 
a  series  of  discharges  from  one  body  to  another  and  where  the  cur- 
rent is  but  of  an  instant's  duration.  The  latter  is  known  as  static 
electricity. 

The  galvanic  cell,  as  usually  constructed,  consists  of  two  metals, 


LABORATORY  MANUAL  OF  PHYSIOLOGY. 

such  as  zinc  and  copper,  partly  immersed  in  a  dilute  acid  or  solu- 
tion of  a  salt.  It  is  now  considered  that  the  current  is  not  due  so 
much  to  the  difference  of  potential  of  the  metals  as  it  is  to  the  dis- 
sociation of  the  solution  into  its  so-called  ions.  These  are  sup- 
posed to  be  charged  with  positive  and  negative  electricity'.  Thus, 
if  HaSOt  be  used  as  the  electrolyte,  the  H  group  represents  ions 
which  are  electropositive,  while  the  SO4  group  is  electronegative. 
These  ions,  charged  with  positive  electricity,  move  toward  the  nega- 
tive pole,  in  this  case,  zinc,  while  those  charged  with  negative  elec- 
tricity, move  toward  the  positive  pole,  in  this  case,  copper.  The 
former  are  termed  kations;  the  latter  are  known  as  anions.  When 
the  twro  metals  are  connected  by  a  conductor,  there  is  a  flow  of 
current  from  the  place  of  highest  intensity,  the  anode,  to  that  of 
lower  intensity,  the  cathode.  This  may  be  compared  to  the  flow 
of  water  from  areas  of  high  pressure  to  areas  of  lower  pressure. 
The  energy  upon  which  the  flow  depends  is  known  as.  the  electro- 
motive force  (E.M.F.).  Its  unit  of  measurement  is  the  VOLT. 

The  flow  of  the  current  meets  with  more  or  less  resistance  which 
has  to  be  overcome.  This  resistance  is  inversely  proportional  to  the 
length  and  thickness  of  the  conducting  substance.  It  also  varies 
with  the  nature  of  the  substance  itself,  irrespective  of  its  length  and 
thickness.  There  is  resistance  in  the  cell  itself.  This  is  known  as 
"internal"  resistance  to  distinguish  it  from  that  in  the  wire  which 
is  known  as  external  resistance. 

Where  the  external  resistance  is  high,  as  in  passage  through  tis- 
sues, the  internal  resistance  of  the  cell  is  a  negligible  quantity. 

2.  Ohm's  Law. — The  relations  between  electromotive  force,  re- 
sistance, and  intensity  of  current,  are  formulated  as  Ohm's  law. 
If  I  represents  intensity  of  current,  E.M.F.,  electromotive  force, 

R 

and  R,  resistance,  then  I  =  - 

E.M.F. 

The  unit  of  measurement  for  the  strength  of  current  is  the  Am- 
pere. This  represents  the  quantity  of  electricity  passing  over  a 
given  cross-section  of  conductor  in  a  given  time.  That  amount  of 
electricity  which  will  deposit  0.001118  grams  of  silver  per  second 

[14] 


MT5CLE-KERVE. 


in  the  cna*  by 

i  r  rif. 

4.  Arrangement  ol  Battery 
pfas  pole,  the  cds 
:•  \-  ":«r   ^    r.r.r::ri 
series.    In  the 

little  change  in  the  voltage.    \\  ith 
is  increased  with  but  &tfe  change  m 


may  be  obtained  bj  joi 
ftam>fisbcd  by  joiuing  gpaaps  of 


Resistance  K>  obtained  by  tibe  use  of  Ike 
ing  of  cofls  of  German-sBvcr  wire 

-.    ,      .      ....  ._  £      ---  ^  _.    1       —    »  -      -         *     i-     •    -   -—  --.     .,.• 

mcuiis  QE  DoctflU  •HOBS  SHBCKPOBB 

of  the  rheocoid,  oonsistnig  of  a  kng  loop  of  the  same 


LABORATORY  MANUAL  OF  PHYSIOLOGY. 


may  be  short-circuited  anywhere  along  its  length  by  means  of  a 
sliding  metal  rider. 

5.  Induced  Currents. — If  a  conductor  through  which  a  current 
is  flowing  is  brought  near  to  another  and  parallel  conductor,  an  in- 
stantaneous current  is  in- 
duced in  the  latter  and  op- 
posite in  direction  to  that  of 
the  former.  When  the  first 
conductor  is  again  removed 
from  the  vicinity  of  the  second, 
a  current  is  again  induced  in 
the  second,  but  now  in  the 
same  direction  as  that  in  the 
first.  The  same  effect  is  pro- 
duced by  making  and  break- 
ing the  circuit  in  the  first  wire 
or  by  alternately  bringing  a 
conductor  into  and  away  from 
the  vicinity  of  a  magnet. 

The  Inductorium,  used  in 
the  physiological  laboratory, 
is  based  on  these  principles 
(see  Fig.  6).  It  consists  of 
a  primary  coil  of  heavier 
wire  of  few  windings  (P),  surrounding  a  movable  core  of 
iron  wire,  a  secondary  coil  (S)  of  finer  wire  and  many  more  wind- 
ings, not  connected  with  the  primary  coil,  and  an  automatic  inter- 
rupter which  may  or  may  not  be  placed  in  circuit  with  the  primary 
coil. 

II.  PRACTICUH. 

Take  two  hollow  spools,  one  considerably  larger  than  the  other, 
winding  the  small  one  with  heavy  insulated  copper  wire  and  the 
large  one  with  fine  insulated  copper  wire.  The  sma1!  coil  is  the 
primary  and  is  to  be  connected  with  the  battery.  The  large  coil  is 
the  secondary  and  is  to  be  connected  with  the  galvanometer. 

[16] 


FIG.  6.— Diagrammatic  Drawing  of  In- 
ductorium. P,  Primary  coil ;  S,  second- 
ary coil ;  C,  core ;  B,  battery  ;  Af,  magnet 
of  interrupter ;  A,  armature  of  interrupter ; 
S#,  spring  of  interrupter. 


MUSCLE-NERVE. 

1.  Place  the  primary  inside  of  the  secondary.    By  means  of  a 
key  interposed  in  the  primary  circuit  (a)  make  the  current.      Is 
there  any  movement  of  the  galvanometer  needle  ?    What  is  the  di- 
rection and  degree  of  this  movement  ?     (b)  Break  the  primary  cir- 
cuit.   What  is  the  movement  of  the  galvanometer  needle?    Does 
the  needle  remain  in  the  position  which  it  assumes  upon  either  the 
make  or  break  of  the  primary  circuit?     (c)  Repeat  (a)  and  (b), 
moving  the  primary  farther  and  farther  from  the  secondary.    Is 
there  any  difference  in  the  excursion  of  the  galvanometer  needle  ? 
Is  there  any  difference  in  the  degree  of  excursion  of  the  needle  at 
make,  as  compared  with  break  of  the  primary  circuit  ? 

2.  (a)  With   the  key  of   the  primary  circuit  closed,  suddenly 
withdraw  the  primary  coil  from  the  secondary.    What  is  the  effect 
on  the  position  of  the  galvanometer  needle?     (b)  Now  suddenly 
approach  the  primary  toward  the  secondary  and  note  the  effect  on 
the  galvanometer. 

3.  (a)  With  the  primary  placed  to  one  side,  introduce  a  per- 
manent magnet  rod  into  the  secondary.    What  is  the  effect  on 
the  galvanometer?     (b)  Withdraw  the  magnet.     What  is  the  re- 
sult? 

4.  With  a  simple  rheocord  in  the  primary  circuit,  suddenly  in- 
crease or  decrease  the  resistance  by  quickly  moving  the  slider  back 
and  forth.     Is  any  current  induced  in  the  secondary  through 
changing  the  intensity  of  the  primary  current  ? 

5.  Apply  the  electrodes  from  the  secondary  coil  of  an  inducto- 
rium  to  the  tip  of  your  tongue.    Open  and  close  the  primary  circuit 
with  the  primary  some  distance  removed  from  the  secondary.    Re- 
peat, gradually  moving  the  secondary  toward  the  primary  until 
the  shocks  produced  are  too  strong  for  comfort.    Which  shock  is 
first  detected  by  the  tongue?    Why  should  the  break  shock  be 
stronger  than  the  make  ?    What  is  the  possibility  of  the  production 
of  induced  currents  in  the  coils  of  the  primary  itself  ?    What  would 
be  their  effect  on  the  make  as  compared  with  the  break  currents  ? 
These  currents  are  known  as  make-extra  and  break-extra  cur- 
rents. 


LABORATORY  MANUAL  OF  PHYSIOLOGY. 

III.  INTERRUPTERS. 

For  interrupting  the  primary  circuit,  any  one  of  a  number  of  de- 
vices may  be  used.  Where  very  rapid  succession  of  shocks  is  de- 
sired, as  in  the  production  of  the  so-called  tetanizing  current,  the 
Neef's  hammer  is  used  as  shown  in  Fig.  6.  Where  known  fre- 


FIG.  7.— Tuning-fork  Interrupter.  (According  to  Kronecker.)  i,  Tuning  fork; 
2,  electro-magnet,  alternately  made  and  broken  ;  3,  battery ;  4,  mercury  contact ;  5, 
time  marker. 

quency  of  interruption  is  necessary,  a  Bowditch  clock  or  similar 
arrangement  may  be  used  for  low  frequencies  and  metronomes  and 
electrically  maintained  tuning  forks,  for  higher  frequencies  (see 
Fig-  7)- 

IV.  DISSECTION  OF  THE  FROG'S  THIGH  AND  LEG. 

With  a  preserved  frog  or  a  fresh  one  that  has  had  brain  and 
cord  destroyed,  carefully  dissect  and  identify  the  muscles  of  the 
thigh  and  leg  (see  Fig.  8). 

1.  Gastrocnemius-sciatic  Preparation. — Pith  a  frog.  Re- 
move the  skin  from  one  thigh.  Make  a  circular  incision  through 
the  skin  at  the  knee  and  another  at  the  lower  end  of  the  leg.  Slide 
this  up  as  far  as  the  knee.  This  is  to  be  slipped  back,  later,  over 
the  muscle  to  keep  it  from  drying.  Separate  the  gastrocnemius 

[18] 


MUSCLE-NERVE. 

muscle  from  the  tibia.  Cut  the  tibia  through,  just  below  the  knee, 
being  careful  to  avoid  injury  to  the  nerve  where  it  enters  the 
muscle  on  its  upper  and  dorsal  aspect.  Tie  a  thread  about  the 


t.a. 


A.  B, 

FIG.  8.— Muscles  of  Frog's  Thigh  and  Leg.  A,  Dorso-lateral  view,  gl,  Gluteus; 
c.e,  coccygeus ;  py,  pyriformis ;  v.e,  vastus  externus;  r.a,  rectus  anterior;  t.f,  triceps 
femoris ;  r.i,  rectus  internus ;  s,  semimembranosus  ;  b,  biceps  ;  g,  gastrocnemius ; 
p,  peroneus;  t.a,  tibialis  anticus.  B,  Ventro-lateral  view,  sar,  Sartorius ;  ad.l, 
adductor  longus  ;  ad.b,  adductor  brevis ;  ad.m,  adductor  magnus  ;  r.i.mi  and  r.i.ma, 
rectus  internus  minor  and  major  (or  gracilis);  g,  gastrocnemius ;  t.a,  tibialis  anticus; 
t.p,  tibialis  posticus. 

tendo  Achillis,  just  above  the  sesamoid  cartilage.     Cut  the  tendon 
below  the  ligature. 

On  the  dorsal  side  of  the  thigh,  carefully  separate  the  gluteus 
maximus  muscle  and  biceps  from  the  semimembranosus,  using  the 
fingers  for  the  purpose.  This  exposes  the  sciatic  nerve.  Carefully 
separate  it  from  the  surrounding  muscles,  avoiding  pulling  and 
stretching  as  much  as  possible.  It  is  well  to  handle  the  nerve  with 


LABORATORY  MANUAL  OF  PHYSIOLOGY. 

a  glass  hook.  Cut  the  thigh  muscles  near  their  insertions  about 
the  knee,  being  careful  not  to  cut  the  nerve.  Cut  the  muscles,  also, 
near  the  pelvic  articulation  of  the  femur.  Remove  all  the  abdom- 
inal viscera,  thus  exposing  the  nerve  in  the  lumbo-sacral  plexus. 
Cut  through  the  vertebral  column  just  above  the  last  two  verte- 
brae which  join  the  urostyle.  Remove 
the  muscles  on  each  side  of  the  urostyle. 
Cut  through  the  junction  of  the  last  ver- 
tebra with  the  urostyle,  and,  using  the 
two  vertebrae  as  a  handle,  lift  the  nerve 
from  its  bed  from  above  downward, 
cutting  its  branches  and  carefully  free- 
ing it  from  the  groove  over  the  femoro- 
pelvic  articulation.  Cut  through  the 
femur  just  below  its  articulation  with 
the  pelvis  and  the  preparation  is  com- 


FIG.  9.-  Gastrocnemius-  sciatic       lt      ^  pj         x        The     femur 
Preparation. 

be  clamped  in  the  muscle  clamp  and  the 

thread  about  the  tendo  Achillis,  to  the  myograph  lever.    Both  nerve 
and  muscle  should  be  kept  moist  with  physiological  salt  solution. 

2.  Double    Semimembranosus-gracilis    Preparation.  —  Dis- 
sect out  the  semimembranosus  and  gracilis  muscles  of  both  sides, 
using    the   same  precautions  as  in  the  previous  preparations. 
Both  femurs  should  be  disarticulated  at  the  hips  and  the  pelvis  cut 
through  transversely,  thus  leaving  the  two  muscles  joined  by  a  thin 
piece  of  bone.    This  may  be  secured  in  the  femur  clamp. 

3.  The  Sartorius.  —  This  corresponds  to  the  muscle  of  the  same 
name  in  human  anatomy.    It  is  a  long,  thin  muscle  having  its  ori- 
gin from  the  symphysis  pubis  and  its  insertion  into  the  capsule  of 
the  knee,  fascia  of  the  leg,  and  tibia.    This  muscle  is  to  be  used 
where  parallel  fibres  are  desired. 

V.  ELASTICITY  or  MUSCLE. 

Make  a  gastrocnemius-muscle  preparation,  clamp  the  femur  in 
the  femur  clamp,  attach  the  tendon  to  the  lever  of  the  myograph, 

[20] 


MUSCLE-NERVE. 

and  adjust  the  writing  point  against  the  smoked  paper  of  a  drum. 
The  lever  should  be  nearly  tangent  to  the  surface  of  the  drum  and 
the  drum  should  revolve  away  from  the  point  of  the  lever.  Ar- 
range the  drum  so  that  it  may  be  revolved  by  hand. 

1.  To  Show  the  Elasticity  of  a  Rubber  Band. — Attach  a  rub- 
ber band  to  the  femur  clamp  and  myograph  lever  and  adjust  for 
writing  on  the  smoked  drum.    Carefully  place  a  lo-gram  weight  in 
the  pan  attached  to  the  lever.  Move  the  drum  slightly  to  record 
the  amount  of  stretching.    Remove  the  weight,  allow  the  lever  to 
return  through  the  elasticity  of  the  band  and  revolve  the  drum 
again  slightly.    Repeat  this  with  20  grams,  with  30  grams,  with  40 
grams,  with  50  grams.    Does  the  lever  return  each  time  to  its  orig- 
inal position  ? 

2.  To  Show  the  Elasticity  of  Muscle.— Repeat  the  above  ex- 
periment, using  the  muscle  already  prepared.    How  does  the  elas- 
ticity of  the  muscle  compare  with  that  of  the  elastic  band  ?    If  the 
elasticity  is  not  perfect  for  the  amount  of  stretching  force  em- 
ployed, are  there  any  factors  of  error  in  the  apparatus  and 
method  that  may,  in  part  at  least,  account  for  the  results  ob- 
tained ? 

3.  To  Show  the  Tensile  Strength  of  Muscle.— With  the  same 
preparation  used  in  the  previous  experiment,  carefully  add  weights 
to  the  pan  of  the  lever  until  something  gives  way.    Which  breaks 
first,  the  muscle  or  the  tendon  ?    Prepare  a  fresh  muscle  and  re- 
peat, inserting  needle  electrodes  into  the  muscle  and  stimulating 
with  a  tetanizing  current  from  an  inductorium  for  each  addition 
of  weight.     How  much  will  the  muscle  lift  ? 

4.  Hooke's  Law. — This  is  embodied  in  the  statement  that  the 
power  of  any  spring  is  in  direct  proportion  to  the  tension  under 
which  it  is  placed.     Does  the  muscle  in  experiment  (b)  respond  to 
Hooke's  law  where  small  weights  are  used  ?    Recent  experiments 
by  Professor  Haycraft,  with  improved  apparatus  from  which  all 
sources  of  error  have  been  eliminated,  show  that,  within  physio- 
logical limits,  all  the  simple  tissues  of  the  body  follow  this  law. 

[21] 


LABORATORY  MANUAL  OF  PHYSIOLOGY. 

VI.  IRRITABILITY  OF  NERVE  AND  MUSCLE  TO 
VARIUS  STIMULI. 

Nerve  and  muscle  tissue  have  in  common  the  properties  of  ir- 
ritability. The  muscle  has,  in  addition,  the  power  of  contractility. 
In  the  following  experiments  the  stimulating  agents  will  be  ap- 
plied to  the  nerve  and  the  contraction  of  the  muscle  will  be  used  as 
an  indicator  of  the  efficacy  of  the  stimulus.  The  stimulating 
agents  may  be  grouped  as  mechanical,  thermal,  chemical,  and 
electrical. 

1.  Mechanical  Stimuli. — Make  a  sciatic-gastrocnemius  prepa- 
ration.   Place  the  nerve  on  a  glass  plate  and  keep  both  muscle  and 
nerve  moist  with  physiological  salt  solution,     (a)  Cut  the  nerve 
near  its  origin  from  the  cord  with  sharp  scissors.    Does  the  muscle 
contract  ?    Tap  the  nerve  with  a  scalpel  handle. 

(b)  Cover  the  nerve  with  moistened  filter  paper,  place  a  thin 
sheet  of  cork  over  this;  place  a  small  beaker  very  carefully  on  the 
cork  and  slowly  pour  mercury  through  a  glass  tube  of  small  calibre 
into  the  beaker.  Does  the  muscle  contract?  Which  is  the  more 
efficacious,  a  stimulating  force  gradually  applied  or  one  suddenly 
applied  ? 

2.  Thermal  Stimuli. — Heat  a  needle  or  a  copper  wire  in  the 
flame  of  a  Bunsen  burner  to  a  red  heat.    Touch  the  nerve  with  it. 
Result?    Will  this  piece  of  nerve  respond  again  to  stimulation? 
Explain. 

3.  Chemical  Stimuli. — Make  a  fresh  muscle-nerve  preparation. 
Cut  the  nerve  high  up.    Place  on  a  glass  plate,  allowing  the  end  of 
the  nerve  to  hang  over  the  edge.    Use  a  watch  glass  or  other  small 
vessel  for  containing  the  reagents  to  be  used.    This  is  brought  up 
to  the  nerve  until  its  end  dips  into  the  contained  reagent.    That 
portion  of  the  nerve  used  is  usually  destroyed  by  the  chemical  so 
that  it  is  necessary  to  cut  off  the  end  of  the  nerve  after  each  test. 
In  this  manner,  try  the  effect  of  the  following  reagents. 

(a)  Concentrated  solution  of  sodium  chloride. 

(b)  Concentrated  solution  of  sodium  or  magnesium  sulphate. 


MUSCLE-NERVE. 

(c)  Fiftv-per-cent  alcohol. 

(d)  Glacial  acetic  acid. 

(e)  Five-per-cent  sulphuric  acid. 
(/)  Ammonia. 

(g)  Zinc  chloride. 

(h)  Allow  the  nerve  to  dry. 

4.  Electrical  Stimuli. — For  this  purpose,  induced  currents  from 
an  inductorium  will  be  used.  The  effect  of  the  constant  current 
will  be  taken  up  later. 

Make  a  muscle-nerve  preparation,  arranging  for  recording  upon 
the  smoked  drum.  Set  up  the  inductorium  for  single  shocks,  in- 
terposing a  short-circuiting  key  in  the  primary  circuit  for  this  pur- 
pose. Remove  the  secondary  coil,  as  far  as  the  instrument  will 
allow,  from  the  primary.  In  those  instruments  where  the  secondary 
is  movable  to  form  angles  of  varying  degrees  with  the  primary,  the 
intensity  of  the  secondary  currents  may  be  diminished  by  increas- 
ing the  angle  between  the  primary  and  secondary  wires. 

Apply  the  electrodes  from  the  secondary  coil  to  the  nerve  or  to 
the  muscle  directly.  Close  the  primary  circuit.  Result  ?  Record 
on  drum.  Rotate  the  drum  slightly  and  break  the  primary  circuit. 
Result  ?  Move  the  secondary  nearer  to  the  primary  and  repeat  the 
make  and  break  as  before,  recording  results.  Repeat  again  and 
again,  gradually  moving  the  secondary  toward  the  primary  until 
the  muscle  ceases  to  increase  in  the  height  of  its  contraction. 
Which  is  the  more  efficacious,  the  make  or  the  break  shock  from 
the  inductorium,  and  why  ? 

VII.  PERIOD  OF  LATENT  STIMULATION  AND  FORM 
OF  THE  SINGLE  TWITCH. 

Arrange  a  drum  to  be  rapidly  spun  by  hand.  With  a  little  prac- 
tice, this  method  gives  as  good  results  as  the  pendulum  or  spring 
myograph.  Arrange  a  muscle-nerve  preparation  with  point  of 
writing  lever  touching  the  smoked  surface  of  the  drum.  Arrange 
the  writing  points  of  two  signal  magnets  (a)  and  (ft),  exactly  under 
the  writing  point  of  the  muscle  lever.  Place  signal  magnet  (a)  in 


LABORATORY  MANUAL  OF  PHYSIOLOGY. 

circuit  with  the  primary.  Place  signal  magnet  (b)  in  circuit  with 
the  tuning  fork  giving  100  interruptions  per  second.  Introduce  a 
short-circuiting  key  in  each  circuit.  Place  the  nerve  of  the  muscle 
preparation  on  the  electrodes  from  the  secondary  coil  of  the  in- 
ductorium. 

The  three  levers,  the  one  attached  to  the  muscle,  that  of  signal 
magnet  (a),  which  is  in  circuit  with  the  current  stimulating  the 
nerve,  and  that  of  signal  magnet  (b),  which  is  in  circuit  with  the 
tuning  fork,  will  then  begin  their  tracings  directly  under  each 
other,  so  that  exact  time  comparisons  may  be  made. 

Close  the  short-circuiting  keys  in  both  circuits.  Start  the  drum 
to  spinning  rapidly.  When  the  drum  has  reached  the  height  of  its 
speed,  open  both  keys.  Close  the  tuning  fork  key  immediately. 
Stop  the  drum.  There  will  be  three  tracings  to  interpret.  The 
signal  magnet  (a)  marks  the  exact  instant  that  the  nerve  was 
stimulated.  The  muscle  lever  marks  the  period  of  contrac- 
tion of  the  muscle.  The  tuning-fork  lever  marks  the  time  re- 
lations. 

Does  the  muscle  twitch  begin  exactly  at  the  moment  of  stimula- 
tion ?  If  not,  how  much  time  elapses  between  the  application  of 
the  stimulus  and  the  onset  of  contraction  ? 

What  is  the  form  of  the  single-twitch  curve  ?  How  does  the  pe- 
riod of  contraction  compare  with  that  of  relaxation  ? 

VIII.  THE  VELOCITY  OF  THE  NERVE  IMPULSE. 

With  the  same  arrangement  of  apparatus  as  in  the  previous  ex- 
periment, ascertain  the  period  of  latent  stimulation  when  the  stim- 
ulating electrodes  are  on  the  nerve  near  the  muscle;  when  the  elec- 
trodes are  on  the  nerve  some  distance  from  the  muscle.  Measure 
the  length  of  nerve  between  the  two  points  of  stimulation.  Esti- 
mate the  difference  in  time  of  latent  stimulation.  This  difference 
will  be  equivalent  to  the  time  that  it  takes  the  nerve  impulse  to 
travel  over  the  length  of  nerve  measured.  From  this  the  velocity 
per  second  can  be  easily  determined. 


MUSCLE-NERVE. 


IX.  DIRECT  IRRITABILITY  OF  MUSCLE;  ACTION  or  CURARE. 

Inject  into  the  dorsal  lymph  sac  of  a  frog  a  few  drops,  about  one- 
half  cubic  centimetre  of  a  one-per-cent  solution  of  curare.  First, 
however,  dissect  out  one  sciatic  nerve,  passing  a  ligature  under  it 
and  tying  it  tightly  about  the  thigh.  All  of  the  frog  except  that  por- 
tion below  the  ligature  will  come 
under  the  influence  of  the  drug 
(see  Fig.  10).  In  fifteen  or  twen- 
ty minutes  the  drug  action  should 
be  complete.  Make  two  sciatic- 
gastrocnemius  preparations,  one 
of  the  curarized  side  and  one  of 
the  non-curarized  side.  Set  up 
the  inductorium  for  tetanizing 
currents.  Attach  muscles  to 
myograph  levers  for  recording. 
Stimulate  nerve  of  curarized  side. 
Result?  Stimulate  the  nerve 
of  the  non-curarized  side.  Re- 
sult ?  Stimulate  the  muscle  of  the 
curarized  side  directly.  Result  ? 

What  is  the  action  of   curare, 
as  deduced  from  these  observations?    Is  the  muscle  fibre  itself 
directly  irritable,  aside  from  its  nerve  ? 

In  the  experiment  under  electrical  stimulation,  it  was  demon- 
strated that,  up  to  a  certain  point,  the  height  of  a  single  muscle 
twitch  is  in  direct  proportion  to  the  strength  of  the  stimulus;  i.e., 
a  minimal  stimulus  is  accompanied  by  a  minimal  contraction  and 
a  maximal  stimulus  by  a  maximal  contraction.  This  will  be  com- 
pared, later,  with  the  action  of  heart  muscle  under  similar  circum- 
stances. 

X.  INFLUENCE  OF  LOAD  ON  MUSCLE  TWITCH. 

When  the  weight  is  continuously  supported  by  the  muscle,  both 
when  at  rest  and  when  contracting,  the  muscle  is  said  to  be  loaded. 


FIG.  10.  —  Curare  Experiment,  i, 
Ligature  around  thigh  ;  j,  sciatic  nerve, 
not  included  in  ligature.  Shaded  area, 
affected  by  curare;  non-shaded  area, 
unaffected  by  curare. 


LABORATORY  MANUAL  OF  PHYSIOLOGY. 

When  the  weight  is  supported  by  the  muscle,  only  during  the  period 
of  contraction,  the  muscle  is  said  to  be  after-loaded.  Compare  the 
muscle  curves  obtained  with  load  and  after-load,  using  first  small 
weights  and  then  heavier  and  heavier  weights. 

XI.  EFFECT  OF  TEMPERATURE  UPON  THE  MUSCLE  CURVE. 

For  the  study  of  the  effects  of  changes  of  temperature  upon  the 
muscle  curve,  the  muscle  warmer  of  Porter  is  very  convenient. 
Where  this  is  not  at  hand,  the  same  results  may  be  obtained  by  the 
use  of  a  bath  of  physiological  salt  solution,  which  may  be  cooled  or 
heated  to  the  desired  temperature  and  in  which  the  muscle  may  be 
immersed,  except  for  the  short  period  required  for  stimulating  and 
recording  contractions. 

1.  Place  the  musde  in  a  small  test  tube  surrounded  by  an  ice 
pack  until  the  temperature  of  the  interior  of  the  tube  has  fallen  to 
zero  or  less,  i.e.,  until  the  freezing  point  has  been  reached.    Re- 
move the  muscle  and  record  a  single  twitch.    Label  the  tracing. 

2.  Warm  the  muscle  to  5°  Centigrade  and  again  record  a  twitch. 

3.  Warm  the  muscle  to  10°  C.  and  again  record. 

4.  Warm  to  15°  C.  and  record  again. 

5.  Warm  to  20°  C.  and  repeat  record. 

6.  Warm  to  30°  C.  and  record  again. 

7.  Warm  to  40°  C.  and  record  again. 

8.  Bathe  the  muscle  with  salt  solution  heated  to  45°  C.  and  note 
result.    The  muscle  passes  into  rigor. 

Compare  the  curves  obtained  at  the  different  temperatures  and 
tabulate  your  results.  WTiat  is  the  effect  on  the  height  of  contrac- 
tion ?  On  the  time  of  the  contraction  phase  ?  On  the  relaxation 
period? 

XII.  INFLUENCE  OF  FATIGUE  ON  THE  FORM  OF  THE  SINGLE 
MUSCLE  TWITCH. 

i.  Set  up  the  apparatus  for  automatic  stimulation  of  the  muscle 
or  nerve  as  shown  in  Fig.  n.  Make  a  sciatic-gastrocnemius 
preparation.  Arrange  the  inductorium  for  maximal  stimulation. 

[26] 


MUSCLE-XERVE. 

Place  the  drum  contact  arrangement  in  circuit  with  the  primary. 
Adjust  the  myograph  lever  to  the  smoked  surface  of  the  drum. 
Allow  the  drum  to  revolve  at  its  greatest  speed.  According  to  the 
arrangement  shown  in  Fig.  n,  six  contractions  will  occur  during 


Jfl 


FIG.  ii.— Drum  Arrangement  for  Muscle-nerve  Stimulation,  d,  Drum,  m,  mus- 
cle ;  »,  nerve ;  e,  electrodes ;  £,  battery ;  »,  inductorium ;  f,  collar  with  contacts 
which,  as  drum  revolves,  make  and  break  primary  circuit  with/",  metal  strip. 

every  revolution  of  the  drum.  Ever}*  sixth  contraction  will  be  re- 
corded at  the  same  place  on  the  drum.  In  this  way  a  number  of 
superimposed  contractions  are  recorded. 

Allow  the  drum  to  revolve  until  the  muscle  ceases  to  respond  by 
a  contraction.  What  changes  does  the  contraction  curve  undergo 
with  the  progression  of  fatigue? 

Compare  the  fatigue  curve  with  the  temperature  curves  ob- 
tained in  the  previous  experiments. 

2.  Make  a  fresh  muscle-nerve  preparation.     Let  the  drum  re- 


LABORATORY  MANUAL  OF  PHYSIOLOGY. 


volve  slowly.  Attach  the  muscle  to  the  myograph  lever.  After- 
load  with  a  lo-gram  weight.  Adjust  lever  to  drum.  Stimulate  the 
nerve  once  a  second  with  a  submaximal  induction  shock. 

A  fatigue  record  formed  of  single  twitches,  written  one  after  the 
other,  will  thus  be  obtained. 

3.  Repeat  the  above  fatigue  experiment  with  a  loaded  muscle. 

XIII.  VOLUME  OF  CONTRACTING  MUSCLE. 

In  answer  to  the  question,  does  the  volume  of  the  muscle  change 
during  contraction,  some  such  device  as  that  shown  in  Fig.  12 
may  be  used.  The  muscle  should  be  put  in  a  container  filled  with 

physiological  salt  solution,  the  ends 
of  the  muscle  being  attached  to  elec- 
trodes from  the  secondary  coil  of  an 
inductorium  arranged  for  tetanizing 
shocks.  The  mouth  of  the  container 
is  closed  with  a  tightly  fitting  cork, 
perforated  for  the  passage  of  a  fine 
glass  tube  in  which  the  water  from 
the  container  rises. 

Stimulate  the  muscle  with  tetaniz- 
ing induction  shocks  and  observe  the 
level  of  the  fluid  in  the  capillary  tube 
connected  with  the  muscle  container. 
Does  the  fluid  rise  or  fall  ?  Does  the 
volume  of  the  muscle  change  during 
contraction  ? 


XIV.  SUMMATION  OF  STIMULI. 


FIG.  12.— To  Determine  Volume 
of  Contracting  Muscle.  Af,  Mus- 
cle ;  e  e,  electrodes ;  c,  capillary 

Arrange  inductorium  with  second- 
ary coil  removed  from  primary  until  single  break  shocks  are  just 
insufficient  to  cause  a  muscle  twitch.  Let  the  muscle  rest  for 
several  minutes.  Now  stimulate  the  nerve  every  four  or  five 
seconds.  Does  the  muscle  finally  contract  ?  Explain. 


[2&] 


MUSCLE-NERVE. 

XV.  SUMMATION  OF  CONTRACTIONS  AND  GENESIS  OF  TETANUS. 

Make  a  semimembranosus-gracilis  muscle  preparation.  Clamp 
bony  attachment  in  the  muscle  clamp.  Attach  the  other  end  to  the 
muscle  lever.  Connect  muscle  up  for  direct  stimulation  with  the 
secondary  coil  of  an  inductorium,  arranged  for  single  shocks.  In 
order  that  the  stimuli  may  be  of  equal  intensity,  it  is  well  to  use 


B 


FIG.  13.— Cut-out  Key.  Bt  Battery ;  P,  primary  coil  of  inductorium ;  S,  secondary 
coil;  a,  &,  and  c,  ct,  metal  strips  ;  i  and  2,  metal  clip  contacts  to  complete  circuit  be- 
tween a,  b,  and  c,  d ;  3,  crank  to  revolve  clip  contacts  i  and  2.  Contacts  revolved  in 
direction  of  arrow.  Contact  at  a,  b,  is  made  an  instant  before  that  at  t,  d.  The 
secondary  circuit  is  therefore  short-circuited  before  the  primary  is  made.  The 
secondary  is  opened  before  the  primary  is  broken.  Hence  the  make  shock  is  cut  out. 
Connect  S  with  c,  d,  and  P  with  a,  t>,  to  cut  out  break  shock. 

some  form  of  cut-out  key,  so  that  either  the  make  or  break  shock 
may  be  eliminated  (see  Fig.  13). 

1.  Arrange  key  to  give  only  break  stimuli.    Stimulate  muscle 
with  one  break  shock.    Note  form  of  contraction  curve  and  height 
of  contraction.     The  drum  should  be  revolving  at  medium  high 
speed. 

2.  Allow  the  muscle  to  rest  for  a  moment.    Stimulate  again,  and, 

[29] 


LABORATORY  MANUAL  OF  PHYSIOLOGY. 

before  the  relaxation  of  the  muscle  is  complete,  send  in  a  second 
stimulus.  Repeat,  decreasing  the  interval  between  stimuli  until  the 
two  contractions  merge  to  form  one.  This  is  known  as  the  sum- 
mation of  contractions.  How  does  the  height  of  the  two  summed- 
up  contractions  compare  with  that  of  the  single  twitch?  Where 
the  second  contraction  begins  during  the  relaxation  phase  of  the 
first,  what  determines  the  height  of  the  second  ? 

3.  Place  the  primary  of  the  inductorium  in  circuit  with  a  metro- 
nome or  vibrating  rod  interrupter.     Stimulate  the  muscle  four 
times  per  second;    five  times  per  second;    six  times  per  second; 
eight  times  per  second;   ten  times  per  second;   twelve  times  per 
second;  fifteen  times  per  second ;  twenty  times  per  second.    Allow 
the  muscle  sufficient  rest  between  the  series  of  stimuli  in  order  to 
avoid  fatigue. 

How  do  the  contraction  curves  change  as  the  frequency  of  stim- 
ulation is  increased  ?  How  many  stimuli  per  second  are  needed  to 
cause  the  individual  curves  to  merge  into  one  apparently  contin- 
uous curve  ?  The  latter  condition  is  called  tetanus.  Where  the  in- 
dividual twitches  are  still  visible,  the  condition  is  one  of  incomplete 
tetanus. 

4.  That  this  condition  of  complete  contraction  is  apparent  rather 
than  real  may  be  shown  by  stimulating  the  nerve  of  a  muscle  and 
connecting  the  muscle  itself  with  a  capillary  electrometer  which 
will  show  a  series  of  action  currents  corresponding  to  a  series  of 
single  twitches.     The  capillary  electrometer  and  the  action  cur- 
rent of  muscle  will  be  described  later. 

5.  Influence  of  Temperature  on  the  Production  of  Tetanus. 
— Repeat  the  previous  experiments  with  a  muscle  cooled  to  5°  C.; 
with  a  muscle  warmed  to  35°  C.    How  does  the  frequency  of  stim- 
ulation necessary  to  produce  tetanus  in  the  cold  muscle  and  in  the 
warm  muscle  compare  with  that  required  for  the  muscle  at  room 
temperature  ? 

6.  Influence  of  Fatigue  on  the  Production  of  Tetanus.— 
After  the  muscle  has  become  tired  from  repeated  stimulation,  re- 
peat the  foregoing  experiments.    Result  ?    Explain. 

[30] 


MTSCLE-KERVE. 

XVI.  To  DETERMINE  ACTUAL  SHORTENING  OF  A  MUSCLE 

LN  CONTRACTION. 

Divide  distance  of  the  writing  point  of  the  muscle  lever  from  the 
axis  of  the  lever  by  the  distance  of  the  muscle  attachment  from  the 
axis.  Then  divide  the  height  of  the  recorded  curve  by  thi>  factor. 
The  result  is  the  actual  shortening  of  the  muscle  during  con- 
traction. 

XVII .  To  DETERMINE  THE  WORK  DONE  BY  THE  MUSCLE  dur- 
ing any  particular  contraction,  multiply  the  actual  shortening  by 
the  load.    Thus,  if  the  actual  shortening  or  height  of  contraction  is 
5  millimetres  and  the  load  is  10  grams,  then  the  work  done  would 
be  50  gram-millimetres, 

1.  On  a  drum  revolved  by  hand,  record  the  heights  of  contrac- 
tion of  a  gastrocnemius  which  is  receiving  submaximal  stimuli. 
After-load  the  muscle  successively  with  10,  20,  30.  40,  50,  70,  100, 
150,  200,  250,  300.  350,  400,  450,  and  500  grams. 

2.  Estimate  the  actual  work  done  according  to  the  formulae 
given  above.    Plot  a  curve,  marking,  on  the  abscissa,  intervals  to 
represent  50-gram  weights;   on  the  ordinates,  intervals  to  repre- 
sent gram-millimetres.    What  conclusions  can  you  draw  from  the 
data  thus  plotted  ? 

XVIII.  FATIGUE  OF  HUMAN  VOLUNTARY  MUSCLE. 

Ergography. — The  contraction  of  voluntary  muscle  is  normally 
brought  about  through  nerve  impulses  originating  in  nerve  cells. 
The  nerve  cell,  as  will  be  shown  later,  has  a  certain  rhythmic  ac- 
tivity, sending  out  from  6  to  10  impulses  per  second.  This  seems 
to  be  sufficient  to  keep  the  muscle  in  a  state  of  tetanus.  The  short- 
est voluntary  muscle  contraction,  then,  brought  about  through  the 
discharge  of  nerve  impulses  from  nerve  cells,  is  a  tetanus.  The 
single  twitch  occurs  only  under  abnormal  circumstances,  or  through 
artificial  stimulation  of  the  nerve  or  muscle  directly.  Any  stimu- 
lation of  nerve  cells,  sufficient  to  cause  a  discharge  of  nerve  im- 
pulses, will  produce  a  tetanus  in  the  muscle  receiving  the  impulses. 


LABORATORY  MANUAL  OF  PHYSIOLOGY 

For  recording  the  contractions  of  human  voluntary  muscle,  either 
the  ergograph  of  Mosso  or  that  of  Porter  may  be  used.  With  the 
former,  the  flexion  of  the  middle  finger  is  recorded ;  with  the  latter 
the  contractions  of  the  abductor  indicis. 

1 .  If  the  Mosso  instrument  is  used,  place  the  forearm  and  fingers 
in  the  securing  attachments  of  the  apparatus  and  weight  the  mid- 
dle finger  with  one  or  two  kilograms.    Contract  the  muscles,  vol- 
untarily, once  every  two  seconds,  keeping  time  to  the  beat  of  a 
metronome,  until  you  are  no  longer  able  to  bring  about  a  contrac- 
tion in  this  way.      The  contractions  should  be  recorded  upon  a 
slowly  revolving  drum.     Now  stimulate  the  flexor  muscles  directly 
with  electrodes  placed  over  the  forearm,  using  the  same  frequency 
of  stimulation  as  before,  one  every  two  seconds.    Does  the  muscle 
respond  to  direct  stimulation  after  fatigue  has  been  induced  to  vo- 
litional impulses  ? 

2.  Repeat  with  a  new  subject,  reversing  the  procedure.     In 
other  words,  stimulate  the  muscle  artificially  until  it  no  longer  re- 
sponds and  then  attempt  to  flex  the  finger  voluntarily  until  com- 
plete fatigue  is  obtained. 

3.  With  a  fresh  subject,  induce  voluntary  fatigue  and  record  the 
time.    Allow  the  muscles  to  rest  for  five  minutes  and  repeat  voli- 
tional contractions  until  fatigue  has  again  occurred.    How  does  the 
time  of  fatigue  onset,  after  the  rest,  compare  with  that  of  the  first 
series  of  contractions  ? 

4.  Now,  instead  of  mere  rest,  give  the  forearm  five  minutes'  mas- 
sage and  repeat  the  ergograph  experiment.    Is  the  onset  of  fatigue 
delayed  as  compared  with  the  first  series  of  contractions,  or  with 
the  second,  or  with  both  ?    What  is  the  effect  of  massage  ? 

XIX.  INFLUENCE  OF  TENSION  ON  THE  MUSCLE  CONTRAC- 
TION.   ISOMETRIC  CONTRACTION. 

In  the  preceding  experiments,  the  resistance  offered  to  the  muscle 
during  its  contraction,  as  measured  by  the  weight  lifted,  has  been 
nearly  uniform,  of  course  excepting  the  inertia  of  the  weight  at  the 
beginning  of  the  lift.  A  muscle  twitch  under  these  circumstances 

[33] 


MUSCLE-NERVE. 

is  called  isotonic.  When  the  shortening  of  the  muscle  is  prevented 
by  a  constantly  increasing  resistance,  so  that  all  its  power  is  used 
in  overcoming  the  resistance,  the  contraction  is  called  isometric. 

The  resistance  is  usually  obtained  by  the  use  of  a  spring  to  which 
the  muscle  is  attached,  the  energy  of  the  muscle  being  stored  in  the 
spring  in  the  form  of  tension,  to  be  liberated  as  heat  as  soon  as  the 
muscle  relaxes. 

1.  Graduation  of  the  Isometric  Spring. — In  order  to  estimate 
the  isometric  value  of  a  muscle  contracting  against  resistance  of- 
fered by  a  spring,  it  is  necessary  to  interpret  the  spring's  resistance 
in  terms  of  weight.     Reverse  the  spring  of  the  heavy  myograph 
(Porter's),  attaching  its  hook  to  the  scale  pan  beneath.     Bring  the 
writing  point  of  the  lever  against  the  smoked  paper  of  a  drum  ar- 
ranged to  be  moved  by  hand.    Revolve  the  drum  a  half  turn  to  re- 
cord a  base  line.    Place  a  loo-gram  weight  in  the  scale  pan.    The 
spring  will  be  bent  to  a  certain  extent  and  the  lever  will  mark  a 
descending  line  on  the  drum.    Move  the  drum,  slightly,  to  record 
the  lower  limit  of  the  spring's  bend.     Repeat  with  a  2oo-gram 
weight,  and  so  on  up  to  800  grams. 

2.  Make  a  gastrocnemius-sciatic  preparation.    Attach  the  tendo 
Achillis  to  the  isometric  spring.     Adjust  the  writing  point  of  the 
lever  against  the  smoked  paper  of  a  rapidly  revolving  drum.    Stim- 
ulate the  nerve  with  a  maximal  break  shock  from  an  inductorium. 
An  isometric  curve  will  thus  be  obtained. 

3.  Release  the  muscle  from  the  spring  and  attach  it  to  the  ordi- 
nary writing  lever  weighted  with  20  grams.    The  lever,  in  this  case, 
should  be  as  long  as  that  used  for  recording  the  isometric  curve. 
With  the  drum  revolving  at  the  same  rate  as  before,  stimulate  the 
nerve  so  as  to  record  a  twitch,  as  nearly  as  possible,  under  the  re- 
corded isometric  curve. 

4.  Compare  the  two  curves  (i)  and  (2),  as  to  form  and  as  to 
work  done.    To  find  the  amount  of  tension  overcome,  as  indicated 
by  weight,  compare  the  height  of  the  isometric  curve  with  the  de- 
pression of  the  spring  in  (i). 

What  influence  does  tension  have  on  muscle  work?    How  does 

3  [33] 


LABORATORY  MANUAL  OF  PHYSIOLOGY. 


this  compare  with  the  muscle  under  isotonic  conditions?  Is  any 
muscle  in  the  body,  normally,  under  isometric  conditions  to  any 
extent  ? 

XX.  ELECTRIC  PHENOMENA  OF  MUSCLE  AND  NERVE. 

1.  Galvani's  Experiment  with  Metals. — Pith  a  frog.     Evis- 
cerate and  remove  everything  above  the  urostyle  and  the  last  two 
vertebrae.    Remove  the  skin  from  both  legs.      Pass  a  hook,  made 
of  clean  copper  wire,  under  both  lumbar  plexuses.     Suspend  the 
preparation  by  the  copper  wire  from  a  clean  iron  or  steel  rod. 
Swing  the  preparation  until  some  part  of  it  comes  in  contact  with 
the  rod.    Result  ?    Explain. 

2.  Contraction  without  Metals. — Make  a  muscle-nerve  prepa- 
ration, cutting  the  nerve  high  up.    Handle  the  nerve  with  a  glass 

hook,  allowing  the  nerve  to 
fall  across  the  muscle.  There 
should  be  a  twitch  of  the  muscle 
every  time  the  nerve  comes  in 
contact  with  it.  This  twitch 
may  be  due  to  one  of  two  things. 
If  the  muscle  is  uninjured,  and 
the  injured  portion  of  the  nerve 
falls  across  it,  the  twitch  may 
be  due  to  the  completion  of  a 
circuit  between  the  injured  and 
uninjured  portion  of  the  nerve 
itself,  which  are  of  different 
electrical  potentials.  Or  it  may 
be  due  to  the  completion  of  a 


FIG.  14.— Secondary  Contraction.  slt 
Sciatic  of  first  preparation ;  J2,  sciatic  of 
second  preparation;  w,,  muscle  of  first 
preparation  ;  »za,  muscle  of  second  prep- 
aration ;  e,  electrodes. 


circuit  between  injured  and  un- 
injured muscle  through  the 
nerve.  This  is  known  as  the  current  of  injury  or  demarcation 
current  of  muscle  or  nerve. 

3.  Secondary  Contraction. — Make  two  muscle-nerve  prepara- 
tions.    Allow  the  nerve  of  preparation  2  (see  Fig.  14)  to  rest  on 

[34] 


MUSCLE-NERVE. 


the  muscle  of  preparation  i.  Stimulate  the  nerve  of  preparatu  n  . 
with  a  tetanizing  current  from  an  inductorium.  Both  muscles  will 
be  thrown  into  tetanus. 

The  nerve  of  the  second  preparation  is  stimulated  by  the  action 
currents  of  the  first  preparation. 

The  action  current  is  due  to  a  change  of  potential  in  an  inactive, 
as  compared  with  an  active  muscle  fibre.  The  same  change  may 
be  demonstrated  in  a  nerve  over  which 
an  impulse  is  passing.  This  may  be 
well  shown  by  means  of  some  form  of 
delicate  current  detector,  such  as  the 
galvanometer  or  capillary  electro- 
meter. 

4.  The  Capillary  Electrometer. — 
This  instrument,  as  commonly  em- 
ployed, consists  of  a  capillary  tube 
containing  mercury  and  dipping  into 
a  vessel  containing  sulphuric  acid. 
The  surface  tension  of  the  mercury  is 
so  great  that  it  does  not  flow  through 
the  fine  capillary  tube,  and  its  upper 
and  lower  meniscus  is  convex  instead 
of  concave  as  is  the  case  with  water. 
The  sulphuric  acid  is  connected  with 
a  platinum  wire.  The  mercury  in  the 
capillary  is  also  supplied  with  a  plati- 
num wire  for  connection  with  any 
source  of  current  (see  Fig.  15).  The 
upper  end  of  the  capillary  tube  is 

connected,  through  a  T  tube,  with  a  mercury  manometer  and  with 
a  pressure  bottle  or  syringe  bulb.  By  raising  the  pressure  bottle  or 
pressing  on  the  bulb,  pressure  is  exerted  upon  the  mercury  in  the 
capillary  tube.  This  pressure  is  measured  by  the  manometer.  By 
pressing  the  mercury  in  the  tube  downward  and  then  releasing  the 
pressure,  some  sulphuric  acid  is  drawn  up  into  the  tube,  into  con- 


FIG.  15.— Capillary  Electrome- 
ter. (Lippmann's.)  e  e,  wires 
leading  to  source  of  current ;  Hg, 
mercury  in  capillary  tube;  Hy 
SO4,  sulphuric  acid. 


LABORATORY  MANUAL  OF  PHYSIOLOGY. 

tact  with  the  mercury.  If  a  current  be  passed  through  the  mercury 
and  sulphuric  acid,  the  surface  tensions  of  the  fluids  are  so  changed 
that  the  mercury  meniscus  will  move  in  the  direction  of  the  current. 
The  extent  of  the  excursion  of  the  mercury  is  in  direct  proportion 
to  the  strength  of  the  current.  This  may  be  measured  by  mercury 
pressure  as  determined  by  the  manometer.  A  fairly  exact  gradu- 
ation of  the  instrument  may  be  made  by  placing  it  in  circuit  with 
currents  of  known  strengths,  and  recording  in  terms  of  mercury 
the  amount  of  pressure  needed  to  bring  the  meniscus  back  to  its 
original  position.  This  instrument  is  exceedingly  sensitive  to  very 
small  currents. 

5.  Current  of  Injury  of  Muscle. — Very  carefully  dissect  out 
the  gracilis  and  semimembranosus  muscles,  avoiding  crushing  or 
tearing  as  much   as  possible.     Place  the  muscle  on   two  non- 
polarizable   electrodes,    connected   by  fine  wire   with  the  galva- 
nometer or  the  capillary  electrometer.     Test  the  non-polarizable 
electrodes  first,  by   bringing  them  in  contact  with  each  other. 
There  should  be  no  deflection  of  the  galvanometer  needle  or  of 
the  mercury  meniscus  of  the  capillary  electrometer. 

(a)  Interpose  a  key  between  the  muscle  and  the  current  de- 
tector.   Close  the  key,  so  that  the  galvanometer  is  brought  in  cir- 
cuit with  the  electrodes  on  the  muscle.    There  should  be  but  little 
if  any  deflection  of  the  needle.    If  the  muscle  were  absolutely  free 
from  injury  and  at  rest,  there  should  be  no  difference  of  potential 
between  any  of  its  parts. 

(b)  Cut  across  one  end  of  the  muscle  with  sharp  scissors  or  scal- 
pel.   Place  one  electrode  on  the  cut  surface,  the  other  on  the  smooth 
surface.    Bring  the  galvanometer  in  circuit  again.    There  is  now 
a  deflection  of  the  needle.    This  is  an  indication  of  the  current  of 
injury  or  the  demarcation  current  of  the  muscle. 

(c)  Cut  off  the  nerve  near  the  muscle  and  repeat  (&),  using  the 
nerve  instead  of  the  muscle. 

6.  Action  Current  of  Muscle. — Make  a  careful  sciatic-gastroc- 
nemius  preparation.    Place  the  muscle  on  the  non-polarizable  elec- 
trodes, connected  with  the  galvanometer.    Place  the  nerve  on  elec- 

[36] 


MUSCLE-NERVE. 


trodes  from  the  secondary  of  an  inductorium  arranged  for  single 
make-and-break  stimuli.  Close  the  galvanometer  key.  There  will, 
probably,  be  more  or  less  deflection  of  the  needle  due  to  the  demar- 
cation current  of  the  muscle  which  was  injured  in  preparation. 
With  the  galvanometer  key  closed,  stimulate  the  nerve  with  a 
single  make  or  break  shock.  The  muscle  will  respond  with  a 
single  twitch.  Is  there  any  movement  of  the  galvanometer  needle  ? 
If  so,  how  much  and  in  what  direction  ? 

7.  Action  Current  of  Frog's  Heart. — Pith  a  frog.     Remove 
the  heart,  being  careful  to  include  the  sinus  venosus.    The  heart 
will,  probably,  continue  its  pulsation 

after  its  removal  from  the  body  of 
the  frog.  Place  the  heart  on  non- 
polarizable  electrodes,  connected 
with  the  capillary  electrometer. 
With  a  low  power  of  the  microscope, 
watch  for  movements  of  the  menis- 
cus of  the  mercury  in  the  capillary 
tube.  How  many  movements  can 
you  make  out  ?  How  do  they  corre- 
spond with  the  beating  of  the  heart  ? 

8.  Paradoxical    Contraction. — 
Pith  a  frog.     Make   a   sciatic-gas- 
trocnemius  preparation,  tracing  out 
and  cutting  the  anterior  tibial  branch 
of  the  nerve.     Stimulate    the    cut 
branch  (see  Fig.  16).     The  muscle 
will  contract. 

9.  Muscle  Tone  of  Rabbit's  Gastrocnemius.     Demonstra- 
tion.—Narcotize  a  fair-sized  rabbit  with  a  hypodermic  injection  of 
one  grain  of  morphine  sulphate.     Complete  anaesthesia  with  ether. 
Tie  rabbit,  belly  down,  on  the  rabbit  board,  with  the  hind  limbs 
well  stretched  out.    Make  a  longitudinal  incision  through  the  skin 
and  separate  the  dorso-lateral  thigh  muscles.     The  large  shiny 
white  sciatic  nerve  will  be  exposed,  deep  in  the  wound.    Tie  a 

[37] 


FIG.  16.  —  Paradoxical  Contrac- 
tion. '  J,  Sciatic  nerve ;  /,  branch 
to  peroneus  muscle ;  e,  electrodes ; 
£•,  gastrocnemius  muscle. 


LABORATORY  MANUAL  OF  PHYSIOLOGY. 

ligature  about  the  nerve  as  high  up  as  possible.  Cut  the  nerve 
above  the  ligature.  Place  the  cut  nerve  on  shielded  electrodes, 
connected  with  the  secondary  coil  of  an  inductorium.  Place  the 
primary  of  the  inductorium  in  circuit  with  a  strong  constant  cur- 
rent interrupted  by  the  tuning-fork  interrupter,  vibrating  one 
hundred  times  per  second.  Place  a  short-circuiting  key  in  the 
secondary  circuit.  Place  the  small  bell  of  a  stethoscope  over  the 
muscle.  Open  the  short-circuiting  key.  The  muscle  will  be 
thrown  into  tetanus  and  the  sound  of  the  vibrating  tuning-fork 
will  be  heard  with  the  stethoscope.  That  this  reproduction  of  the 
tuning-fork  tone  is  really  due  to  the  vibration  of  the  muscle  fibres 
to  each  individual  stimulus  from  the  inductorium  is  shown  by 
the  next  experiment. 

10.  Action  Currents.  Detection  of,  with  the  Telephone.— 
With  the  same  preparation  as  in  the  previous  experiment,  insert 
needle  electrodes  into  the  body  and  tendinous  portion  of  the  gas- 
trocnemius  muscle.  Connect  these  with  a  telephone  receiver  and 
again  stimulate  the  sciatic  with  one  hundred  shocks  per  second. 
You  will  now  hear  the  sound  of  the  tuning-fork  reproduced  in 
the  telephone.  This  is  due  to  the  development  of  action  currents 
in  the  muscle  corresponding,  in  frequency,  to  the  number  of  im- 
pulses coming  to  the  muscle. 

This  sound  is  known  as  the  artificial  muscle  tone,  to  distinguish 
it  from  the  muscle  sound  which  occurs  when  the  muscle  is  con- 
tracted under  the  influence  of  volition  and  which  is  called  the  nat- 
ural muscle  tone. 

This  may  be  heard  by  placing  the  stethoscope  on  the  biceps 
muscle  and  strongly  flexing  the  forearm  on  the  arm. 

XXI.  IRRITABILITY  AND  CONDUCTIVITY  OF  NERVE  AND  MUSCLE 
DURING  AND  AFTER  THE  PASSAGE  OF  A  CONSTANT  CUR- 
RENT. ELECTROTONUS. 

During  the  passage  of  a  constant  current  through  a  nerve,  the 
irritability  and  conductivity  are  increased  at  the  kathode,  where 
the  current  leaves  the  nerve,  and  diminished  at  the  anode,  where 

[38] 


MUSCLE-NERVE. 

the  current  enters  the  nerve.  Immediately  after  the  cessation  of 
the  current,  these  conditions  are  reversed ;  the  irritability  and  con- 
ductivity are  increased  at  the  anode  and  decreased  at  the  kathode. 


\ 


FIG.  ^.-Arrangement  for  Studying  Effect  of  Constant  Current  on  Irritability  of 
Nerve.  Current  running  in  direction  of  arrow  is  a  descending  current.  Inductoria 
(i  and  2)  connected  with  battery  through  current  changer  (3)  in  such  a  way  that  cur- 
rent may  be  passed  through  primaries  of  either  i  or  2,  so  as  to  stimulate  nerve  (4)  m 
region  of  anelectrotonus,  about  anode  (6),  or  in  regions  of  kathelectrotonus,  about 
kathode  (8).  Nerve  impulse  indicated  by  twitch  of  muscle  (5).  Through  Pohl's 
commutator  (7),  with  crosspieces  in,  the  constant  current  maybe  reversed  and  be- 
come an  ascending  current,  instead  of  a  descending  as  shown  in  the  figure.  The 
dotted  line,  running  below  the  nerve  at  the  anode  and  above  at  the  kathode,  repre- 
sents, respectively,  the  diminution  and  increase  in  irritability  of  nerve  in  the  anodic 
and  kathodic  regions. 

[39] 


LABORATORY  MANUAL  OF  PHYSIOLOGY 

This  condition  of  change  in  a  nerve  or  a  muscle,  since  the  muscle 
itself  reacts  in  the  same  manner  as  the  nerve,  is  known  as  electro- 
tonus.  The  condition  of  the  nerve  about  the  anode  is  called  anelec- 
trotonus;  that  about  the  kathode  is  called  katelectrotonus.  The 
conditions  of  anelectrotonus  and  katelectrotonus  are  most  marked 
in  the  immediate  vicinity  of  the  anode  and  kathode.  From  these 
poles  they  gradually  diminish  in  the  extrapolar  and  interpolar 
regions,  until,  in  the  latter,  a  neutral  point  is  reached  about  midway 
between  the  two  poles  (see  Fig.  17). 

When  the  kathode  is  near  the  muscle  and  the  anode  farther  from 
the  muscle,  the  current  is  said  to  be  descending.  When  these  con- 
ditions are  reversed,  that  is,  when  the  anode  is  near  the  muscle,  the 
current  is  said  to  be  ascending.  In  the  following  experiments  the 
electrotonic  conditions  will  be  tested  with  the  nerve,  the  muscle 
twitch  being  used  as  a  convenient  indicator. 

i.  Make  a  sciatic-gastrocnemius  preparation.  Save  the  whole 
length  of  the  nerve.  Arrange  moist  chamber  with  non-polarizable 
electrodes  placed  in  circuit  with  one  or  more  battery  cells,  rheo- 
cord,  and  Pohl's  commutator  or  current-changer.  Place  the  nerve 
upon  the  non-polarizable  electrodes  as  shown  in  Fig.  17.  Place 
a  pair  of  platinum  electrodes  from  the  secondary  coil  of  an  induc- 
torium  on  the  nerve  at  the  anode  of  the  constant  current,  and  an- 
other at  the  kathode  of  the  constant  current.  Arrange  as  in  Fig. 
17,  so  that  the  nerve  may  be  stimulated  with  the  induced  current 
at  either  pole  of  the  constant  current.  By  means  of  the  Pohl's 
commutator,  the  constant  current  may  be  reversed  in  direc- 
tion; i.e.,  it  may  be  made  either  an  ascending  or  a  descending 
current. 

Attach  the  tendon  of  the  muscle  to  the  writing  lever  of  a  myo- 
graph.  Let  this  record  the  contractions  of  the  muscle  on  a  drum 
arranged  to  be  revolved  by  hand.  Send  an  ascending  current 
through  the  nerve.  While  the  constant  curfent  is  passing,  stimu- 
late the  nerve  in  the  anodic  region  with  a  medium  strong  single- 
break  shock  from  the  inductorium.  Mark  result  of  muscular  con- 
traction, if  any,  on  drum,  as  well  as  data  needed  to  identify  what 

[40] 


MUSCLE-NERVE. 

you  have  done.  Revolve  drum  a  sufficient  distance  and  repeat, 
stimulating,  this  time,  in  the  kathodic  region.  Record  data  as 
before. 

2.  Now,  reverse  the  constant  current  so  that  the  anode  is  away 
from  the  muscle  and  the  kathode  is  near  the  muscle.    Repeat  the 
stimulation  with  the  induced  current  in  the  anodic  and  kathodic 
regions,  as  before.    Record  the  muscle  response,  or  the  lack  of  it, 
on  the  drum  and  make  careful  note  of  all  the  data. 

From  these  experiments,  what  conclusions  can  you  draw  con- 
cerning the  effect  of  the  passage  of  a  constant  current  upon  the 
irritability  of  a  nerve  ? 

3.  Repeat  the  above  experiments  with  ascending  and  descend- 
ing currents,  stimulating  in  the  anodic  and  kathodic  regions  im- 
mediately after  the  cessation  of  the  constant  current.    What  is  the 
after-effect  of  the  passage  of  the  constant  current  upon  the  irrita- 
bility of  the  nerve  ? 

XXII.  THE  CONSTANT  CURRENT  AS  A  STIMULUS.    PFLU 
GER'S  LAWS. 

As  we  have  already  seen,  a  sudden  increase  in  intensity  of  a  stim- 
ulus which  is  being  applied  to  a  nerve  or  a  muscle  is  effective  in 
producing  an  impulse  in  the  same.  A  gradual  increase,  on  the 
other  hand,  is  not  effective. 

In  the  same  way  a  sudden  increase  in  irritability  will,  of  itself, 
act  as  a  stimulus.  Thus,  when  a  constant  current  of  sufficient 
strength  is  passed  through  a  nerve,  there  is  a  sudden  increase  in 
irritability  in  the  region  of  the  kathode  and  a  sudden  decrease  in 
irritability  at  the  anode.  There  will  then  be  a  stimulation  of  the 
nerve  in  the  kathodic  region  which  will  cause  an  impulse  to  be 
transmitted  toward  the  muscle.  If  the  current  is  an  ascending  one, 
however,  and  the  conductivity  is  sufficiently  diminished  at  the 
anode,  the  impulse  will  be  blocked  and  the  muscle  will  not  respond 
with  a  contraction. 

When  the  constant  current  ceases  to  flow,  the  irritability  sud- 
denly falls  at  the  kathode  and  rises  at  the  anode.  The  conductivity 

[41] 


LABORATORY  MANUAL  OF  PHYSIOLOGY. 


changes  likewise,  but  not  necessarily  to  the  same  degree.  If  the 
current  is  ascending  and  the  increase  in  irritability  is  sufficient,  the 
muscle  will  respond  with  a  contraction.  If  the  current  is  descend- 
ing, and  the  fall  in  conductivity  at  the  kathode  is  sufficient,  this 
will  act  as  a  block  between  the  anode  and  the  muscle  and  no  con- 
traction will  be  obtained.  The  relative  increase  and  decrease  of 
irritability  and  conductivity  at  the  anode  and  kathode  vary  with 
the  strength  of  the  contsant  current  employed.  Other  things  being 
equal,  the  make  of  the  constant  current  is  more  efficacious  as  a 
stimulus  than  the  break. 

These  facts  have  been  formulated  as  Pfluger's  laws.    Briefly, 
they  are  as  follows: 


Strength. 

Direction  of  Current. 

Ascending. 

Descending. 

Weak 

Make. 
No 
Yes 
Yes 
No 

Break. 
No 
No 
Yes 
Yes 

Make. 
Yes 
Yes 
Yes 
Yes 

Break. 
No 
No 
Yes 

No 

Medium  
Medium  strong 
Strong  

The  words  "yes"  and  "no"  in  the  above  table  indicate  the  oc- 
currence or  absence  of  a  muscle  contraction  under  the  circum- 
stances noted.  The  strength  of  the  current  is  regulated  by  increas- 
ing or  decreasing  the  resistance  by  means  of  a  rheocord  or  resist- 
ance box. 

With  a  fresh  muscle-nerve  preparation,  make  and  break  a  con- 
stant current  through  the  nerve  as  indicated  by  the  above  table. 
In  the  light  of  the  explanations  which  have  been  given  and  the 
previous  experiments  performed,  how  are  these  results  to  be  ex- 
plained ? 

XXIII.  STIMULATION  OF  HUMAN  NERVES. 

In  human  nerves,  in  the  body  during  life,  it  is  obviously  imprac- 
ticable to  bring  the  electrodes  into  direct  contact  with  the  nerve. 
There  is  more  or  less  insulation  from  the  intervening  integument 

[4*] 


MUSCLE-NERVE. 

and  fat.  In  this  case  the  electrode,  anode  or  kathode,  is  brought 
into  as  close  relation  to  the  nerve  as  possible.  Only  a  small  por- 
tion of  the  current  will  traverse  the  nerve,  longitudinally.  The 
greater  part  of  the  current  will  traverse  the  nerve  diagonally,  form- 
ing current  loops  which  spread  through  the  tissues,  finally  concen- 
trating to  pass  through  the  nerve  again  to  the  other  electrode. 

Those  points  at  which  the  current  enters  the  nerve  are  known  as 
physiological  anodes,  and  those  where  the  current  leaves  the  nerve, 
as  physiological  kathodes.  Thus,  at  each  pole  groups  of  physio- 
logical anodes  and  kathodes  are  found.  The  contraction  of  the 
muscle  which  occurs  when  the  current  is  closed  represents  irritation 
at  the  physiological  kathode.  That  contraction  occurring  at  the 
break  of  the  current  represents  irritation  at  the  physiological 
anode. 

Since  there  are  both  physiological  anodes  and  kathodes  at  each 
pole,  any  one  or  more  of  the  following  results  may  be  obtained 
through  the  opening  or  closing  of  the  constant  current: 

1.  Anodic  Closing  Contraction. — Contraction   following  the 
change  developed  at  the  physiological  kathode  beneath  the  phys- 
ical anode. 

2.  Anodic  Opening  Contraction. — Contraction  following  the 
change  produced  in  the  nerve  at  the  physiological  anode  beneath 
the  physical  kathode. 

3.  Kathodic  Closing  Contraction. — Contraction  following  the 
change  produced  in  the  nerve  at  the  physiological  kathode  beneath 
the  physical  kathode. 

4.  Kathodic   Opening   Contraction. — Contraction    following 
the  change  produced  in  the  nerve  at  the  physiological  anode  be- 
neath the  physical  kathode. 

The  following  abbreviations  for  these  contractions,  from  i  to  4 
respectively,  are  used:  ACC,  AOC,  KCC,  KOC. 

KCC  and  ACC  are  stronger  than  KOC  and  AOC.  KCC  is 
stronger  than  ACC,  and  AOC  is  stronger  than  KOC. 

The  effect  of  change  of  strength  of  current  is  shown  in  the  fol- 
lowing table: 

[43] 


LABORATORY  MANUAL  OF  PHYSIOLOGY. 


Weak  Currents. 

Medium  Currents. 

Strong  Currents. 

KCC 

KCC 

Arc 

KCC 

Arr 

Aor 

AOP 

"K"OP 

With  a  strong  current,  tetanus  sometimes  occurs  both  at  the 
make  and  the  break. 

1.  Experiment. — Set  up  8  or  10  dry  cells  in  series.    Place  a 
commutator  and  simple  key  in  circuit  with  the  brass  electrodes  to 
be  used  for  the  stimulation  of  the  human  nerve.    Place  the  anode 
electrode  on  the  back  of  the  neck  and  the  kathode  over  the  ulnar 
nerve  at  the  elbow. 

(a)  Make  and  break  the  circuit  with  the  simple  key.     If  there 
is  no  accompanying  muscular  contraction,  add  more  cells  to  the 
circuit  until  a  contraction  is  obtained.     Record  results  and  com- 
pare with  the  table  given  above. 

(b)  Change  the  direction  of  the  current  by  means  of  the  com- 
mutator key  so  as  to  make  the  electrode,  over  the  nerve,  the  anode. 

Again  make  and  break  the  circuit,  starting  with  weak  currents 
and  increasing  the  strength  of  current  as  before.  Keep  a  careful 
record  of  the  various  strengths  of  current  and  the  appearance  of 
the  different  contraction  responses. 

2.  Reaction  of  Degeneration. — In  a  muscle  whose  nerve  has 
been  cut  off  from  its  controlling  cell,  after  a  time  certain  definite 
changes  in  irritability  to  the  constant  and  induced  currents  occur. 
There  is  a  gradual  diminution  in  excitability  to  the  induced  cur- 
rent and  at  first  an  increased  excitability  to  the  constant  current. 
Later,  this  diminishes  also.    The  muscle  contraction  may  also  be- 
come greatly  prolonged  and  a  condition  called  galvanotonus  (tonic 
contraction)  may  be  easily  produced.     The  normal  contraction 
formula  is  departed  from,  the  most  characteristic  change  being  a 
reversal  of  the  usual  appearance  of  KCC  and  ACC.    Normally, 

[44] 


MUSCLE-NERVE. 

KCC  appears  before  ACC.    In  well-marked  degeneration  ACC 
appears  first. 

Experiment. — Narcotize  a  rabbit  with  morphine  and  ether.  Ex- 
pose and  cut  one  sciatic  nerve  as  high  up  as  possible.  Sew  up  the 
wound  and  test  the  muscle  at  intervals  of  two  days  for  the  reaction 
of  degeneration. 

XXIV.  ACTION  OF  CERTAIN  DRUGS  UPON  THE  SINGLE 
MUSCLE  TWITCH. 

1.  Veratrine. — Make  a  saturated  solution  of  veratrine  in  0.6- 
per-cent  sodium-chloride  solution.    Pith  a  frog  and  inject,  under 
the  skin,  about  five  drops  of  the  veratrine  solution.    In  a  few  min- 
utes (ten  to  fifteen),  if  the  frog  be  made  to  jump,  it  will  be  seen 
that  the  recovery  of  the  flexed  position  of  the  hind  limbs  is  very 
slowly  brought  about.     A  little  later,   a  spasm  of  both  limbs 
occurs  at  every  attempt  to  jump.     The  flexor  muscles,  being  the 
weaker,  are  overpowered  by  the  extensors. 

Make  a  gastrocnemius-sciatic  preparation  from  the  frog.  At- 
tach the  tendon  to  the  writing-lever  of  a  myograph.  Adjust  the 
point  of  the  lever  to  the  smoked  paper  of  a  drum  revolving  at  me- 
dium speed.  Stimulate  the  nerve  with  a  single  maximal  break 
shock  from  an  inductorium.  How  does  the  recorded  contraction 
compare  with  the  normal  muscle  twitch  ? 

2.  Adrenalin    (active  principle  of  the  suprarenal  gland). — In- 
ject into  the  dorsal  lymph  sac  of  a  pithed  frog  10  drops  of  a  i  to 
10,000  solution  of  adrenalin  chloride.    After  ten  minutes,  make 
a  muscle  preparation  and  proceed  as  in  i .    Is  there  any  departure 
from  the  normal  muscle  twitch  ?    Prepare  the  muscle  and  nerve  of 
the  other  leg  or  of  another  frog.     Immerse  the  muscle  in  the 
adrenalin  for  a  few  minutes  and  again  record  a  contraction. 

XXV.  INVOLUNTARY  MUSCLE. 

Synonymes :  Plain  or  smooth,  slow,  non-striated,  i .  Remove  the 
stomach  from  a  pithed  frog.  Make  two  parallel  cuts  through  the 
viscus  running  at  right  angles  to  the  long  axis  and  about  one-half 

[45] 


LABORATORY  MANUAL  OF  PHYSIOLOGY. 

centimetre  apart.  Pass  a  bent  pin  through  the  ring  thus  made,  and 
support  this  in  the  femur  clamp  of  the  myograph.  Pass  a  fine 
copper  wire  through  the  lower  portion  of  the  muscle  ring  and  at- 
tach it  to  the  muscle  lever  and  the  binding  post  of  the  lever.  Con- 
nect this  post  and  that  of  the  femur  clamp  with  a  dry  cell,  inter- 
posing a  simple  key  and  a  signal  magnet  in  the  circuit.  Adjust  the 
point  of  the  muscle  lever,  the  point  of  the  signal  magnet,  and  the 
point  of  a  chronograph  lever,  marking  hundredths  of  a  second,  in 
a  straight  perpendicular  line  against  the  smoked  paper  of  a  drum 
arranged  to  revolve  at  high  speed.  Start  the  drum,  place  the  chro- 
nograph in  circuit  with  the  vibrating  tuning-fork,  open  and  close 
the  key  in  the  battery  circuit. 

Compare  the  length  of  the  latent  period  and  the  form  of  the  con- 
traction curve  with  that  of  skeletal  muscle. 

2.  Make  a  second  similar  preparation,  omitting  the  electrical 
apparatus  for  stimulation,  and  allow  the  lever  of  the  myograph  to 
rest  against  a  drum  revolving  once  an  hour. 

Observe  tonic-  contractions  of  the  muscle. 


i46] 


CHAPTER  III. 


NERVOUS  SYSTEM. 

I.  REFLEX  ACTION. 

CHLOROFORM  a  frog.  Make  a  longitudinal  incision  through 
the  skin  in  the  middle  line  of  the  skull.  Cross  this  with  another 
incision  from  ear  drum  to  ear  drum.  Turn 
back  the  skin  flaps  and  expose  the  skull. 
Carefully  remove  this  piecemeal  with  strong 
scissors  and  forceps  from  before  backwards. 
Expose  the  brain,  noting  its  relations  to  the 
landmarks  on  the  skull.  Compare  with 
Fig.  18. 

1.  Reflex  Action  with  Cerebrum  only 
Removed. — Partly  anaesthetize  a  frog.  Cut 
through  the  skull  with  sharp  scissors  or 
scalpel,  transversely,  just  in  front  of  the  ear 
drums.  This  will  serve  to  eliminate  the 
cerebral  lobes.  Clamp  the  lower  jaw  in  a 
femur  clamp  and  suspend  the  frog  from  an 
upright  stand.  Keep  the  wound  made,  moist 
with  physiological  salt  solution.  Allow  the 
frog  time  to  recover  from  the  shock  of  the 
operation  and  try  the  following  experiments: 

(a)  Immerse  one  foot  in  a  beaker  contain- 
ing a  dilute  solution  of  sulphuric  acid  (i  to 
10,000).     Note  the  time  elapsing  between  the 
application  of  the  stimulus  and  the  first  muscular  contraction. 
Which  muscles  contract  first?    Does  the  reaction  extend  to  any 

[47] 


9 


FIG.  18.  —  Frog's 
Brain .  i,  Olfactory 
nerves;  2,  olfactory 
lobes ;  3,  cerebral  lobes ; 
4,  epiphysis  cerebri 
(pineal  body)  ;  5,  optic 
thalamus ;  6,  optic 
lobes ;  7,  cerebellum ;  8, 
medulla ;  9,  rhomboid 
fossa  (fourth  ventricle). 


LABORATORY  MANUAL  OF  PHYSIOLOGY. 

other  muscles  if  the  application  of  the  stimulus  is  continued? 
Explain. 

(b)  Wash  the  foot  thoroughly  in  plain  tap  water  and  dry  with 
filter  paper.    Repeat  experiment  (a),  using  a  stronger  solution  of 
the  acid  (i  to  1000).    Record  reflex  time  as  before. 

(c)  Wash  the  feet  again  and  dry  with  filter  paper.     Repeat  ob- 
servations, using  a  still  stronger  solution  of  acid  (i  to  500),  and  re- 
cord results.    What  is  the  effect  on  reflex  time  of  increasing  the 
strength  of  the  stimulus  ? 

(d)  Instead  of  the  acid,  use  medium  make-and-break  shocks 
from  an  inductorium   as    the   stimulating    agent.      Make    and 
break  every  three  seconds  for  ten  shocks.     Is  there  any  reflex 
response  ? 

(e)  Increase  the  frequency  of  stimulation  to  one  per  second;  to 
two  per  second.    Is  there  any  reflex?    If  so,  how  many  stimuli 
must  be  applied  to  the  skin  before  the  reflex  arc  is  completed? 

>  Where  are  the  main  places  of  resistance  in  the  reflex  arc  ? 
i  2.  Reflex  Action  with  Optic  Lobes  also  Removed.— Using 
the  same  frog  as  in  the  previous  experiments,  make  an  incision 
through  the  skull  and  brain  just  behind  the  tympanic  membrane. 
Allow  time  for  the  nervous  system  to  recover  from  the  shock  of  the 
operation  before  proceeding  with  the  next  series  of  observations. 

Repeat  experiments  (a)  to  (e)  of  series  i.  How  does  the  reflex 
time  of  this  set  of  experiments  compare  with  that  of  the  previous 
set? 

What  is  your  conclusion  concerning  the  influence  of  the  optic 
lobes,  in  the  frog,  on  cord  reflexes  ? 

3.  Reflex  Action  with  Medulla  Removed. — Complete  the 
pithing  of  the  frog  and  repeat  the  previous  series  of  experiments. 
Conclusions? 

J  4.  Diffusion  of  Impulses  within  the  Cord. — Pith  another 
frog.  Suspend  as  before.  Apply  a  strong  and  continuous  stimulus 
to  one  foot.  Note  the  successive  groups  of  muscles  that  become 
involved  in  the  reflex  reaction.  Also  note  the  time  at  which  each 
group  becomes  involved  and  the  order  of  response. 

[48] 


NERVOUS  SYSTEM. 

6.  Apparent  Purposive  Character  of  Reflex  Responses. — 

A  pithed  frog  is  prepared  as  in  the  previous  experiments.  Take 
a  small  piece  of  filter  paper  wet  with  acetic  acid  diluted  one-half 
with  water,  and  apply  this  to  the  ventral  aspect  of  one  thigh.  Note 
the  attempt  to  remove  this  with  the  foot  of  the  same  side.  If  this 
is  unsuccessful,  or  if  the  leg  be  held  fast,  the  foot  of  the  opposite 
side  will  be  brought  into  play  and  even  the  fore  limbs,  in  an  attempt 
to  brush  off  the  offending  irritant. 

6.  To  Show  the  Centres  of   Reflex  Exchange  in  the  De- 
cerebrized  Frog. — Using  the  frog  of  the  previous  experiment, 
run  a  long  needle  through  the  neural  canal  to  destroy  the  spinal  cord. 
After  a  sufficient  interval,  test  the  frog  as  before  for  reflexes.   Result  ? 
What  is  the  function  of  the  cord  in  relatioli  to  reflex  action? 

Test  the  excitability  of  the  muscles  and  nerves  with  the  induced 
current.  Open  the  thorax  and  observe  the  beating  of  the  heart. 
The  frog  is  still  alive  so  far  as  the  vegetative  functions  are  con- 
cerned, but  the  entire  cerebro-spinal  axis  is  destroyed  and  conse- 
quently all  reflex  action  is  abolished. 

7.  Action  of  Strychnine. — Decerebrize  a  frog.     Test  the  re- 
flexes as  in  experiment  2.     Now  inject  under  the  skin  a  solution 
containing  one-half  milligram  of  strychnine  sulphate.     Test  re- 
flexes again,  as  before,  at  five-minute  intervals.     If  there  is  no  ap- 
preciable change  after  ten  minutes,  repeat  the  injection.     What 
is  the  effect  upon  reflex  time  as  compared  with  the  normal  for 
the  frog  used  ? 

Repeat  the  injection  until  the  frog  is  thrown  into  tetanic  spasms 
upon  the  slightest  stimulation.  What  is  the  character  of  these 
spasms?  What  is  the  position  of  the  frog  during  a  convulsion? 
Explain. 

Now  destroy  the  cord  by  passing  a  needle  through  the  neural 
canal.  What  is  the  effect  upon  the  strychnine  spasms  ?  What  is 
the  seat  of  the  strychnine  action  ? 

8.  Action  of  Chloral  Hydrate. — Pith   another   frog.     Estab- 
lish the  normal  reflex  time  to  some  stimulus,  taken  as  a  stand- 
ard.   Inject  under  the  skin  10  drops  (about  0.6  c.c.)  of  a  2-per- 

4  [49] 


LABORATORY  MANUAL  OF  PHYSIOLOGY. 

cent  solution  of  chloral  hydrate.  Test  the  reflexes  as  before  and 
compare  with  the  normal  taken  as  a  standard.  How  does  the  effect 
of  the  chloral  compare  with  that  of  the  strychnine?  Repeat  the 
injection  of  the  chloral  until  a  lethal  effect  is  produced.  Note  all 
accompanying  phenomena. 

II.  REACTION  TIME. 

1.  For  Sound. — A  tuning  fork  vibrating  one  hundred  times  per 
second  is  placed  in  circuit  with  a  time-marker.     Two  short-cir- 
cuiting keys  are  placed  in  circuit  with  the  time-marker,  so  that,  by 
closing  either  one,  the  time-marker  may  be  cut  out  of  the  circuit. 
The  student  experimented  upon  holds  the  handle  of  one  key.    An- 
other student  holds  the  handle  of  the  other  key.    The  first  key  is 
held  open,  the  second  key  being  closed.    Let  the  subject  of  the  ex- 
periment close  his  eyes.    He  should  close  his  key  as  soon  as  he 
hears  the  opening  click  of  the  other  key. 

The»opening  of  the  one  key  sets  the  time-marker  to  recording. 
The  closure  of  the  other  key  stops  the  time  record.  This  should 
be  taken  upon  a  rapidly  revolving  drum.  The  interval  between 
the  opening  of  the  circuit  and  its  closure  is  marked  in  hundredths 
of  a  second  and  represents  the  time  occupied  for  the  passage  of  the 
sound  wave  through  the  auditory  apparatus,  the  auditory  nerve  to 
the  auditory  centres  of  consciousness;  its  transfer  to  a  motor 
neuron;  its  passage  to  the  muscles  involved  and  the  latent  period 
of  these  muscles. 

Repeat  the  experiment  ten  times  for  each  individual  and  try 
a  number  of  different  individuals.  Estimate  the  average  reaction 
time  for  each  individual. 

2.  For  Vision. — For  the  first  key  to  make  the  circuit,  use  a 
mercury  contact.    Darken  the  room,  so  that  the  spark  made  when 
the  key  is  opened  may  be  distinctly  seen.    Let  the  subject  of  the 
experiment  close  his  key  as  soon  as  he  sees  the  spark  made  by  the 
opening  of  the  other.     The  interval  marked  by  the  tuning  fork 
gives  the  reaction  time  for  vision.     This  experiment  should  also 
be  repeated  ten  times  and  the  average  taken. 


NERVOUS  SYSTEM. 

3.  For  Tactile  Sensation. — The  circuit-opening  key  may  be 
so  arranged  that,  upon  opening,  it  will  come  into  sharp  contact 
with  the  finger  of  the  subject  who  is  to  close  his  key  as  soon  as  he 
feels  this  contact.     This  will  give  the  reaction  time  for  tactile 
sensation. 

4.  Influence   of   Drugs    on   the   Reaction    Time. — Repeat 
experiments  i  to  3  on  one  of  the  subjects  whose  normal  reaction 
time  has  already    been   determined,   after  drinking  30  c.c.   of 
whiskey  diluted  with  equal  parts  of  water. 

III.  REMOVAL  OF  CEREBRUM  IN  THE  FROG. 

Remove  the  cerebral  hemispheres  in  a  frog  by  making  a  cut 
through  the  skull  in  front  of  the  tympanic  membranes.    Compare 

t  2 


FIG.  19.— Pigeon's  Brain,    i,  Skull  which  has  been  removed  in  part  to  show  relations 
of  cerebrum  1^2),  cerebellum  (3),  and  medulla  (4). 

the  frog  so  treated  with  a  normal  frog.  Place  both  the  normal  and 
the  decerebrized  frogs  on  their  backs.  How  do  they  react  ?  Stim- 
ulate the  decerebrized  frog.  Is  there  any  change  in  the  co-ordina- 
tion of  movements?  Place  both  frogs  in  water.  Can  the  decere- 
brized frog  swim  in  a  normal  manner? 

Make  cut  behind  the  tympanic '  membranes.     Compare  this 
condition  with  the  preceding  and  with  the  normal. 

[51] 


LABORATORY  MANUAL  OF  PHYSIOLOGY. 

IV.  REMOVAL  or  THE  CEREBRUM  IN  A  PIGEON. 

Remove  the  cerebral  hemispheres  in  a  pigeon  anaesthetized  with 
ether.  Sew  skin  over  wound.  Allow  the  pigeon  twenty-four  hours 
to  recover  from  the  effect  of  the  operation.  Compare  the  pigeon 
so  treated  with  a  normal  bird.  Is  there  any  disturbance  of  co-or- 
dination ?  Is  the  pigeon  able  to  sit  on  a  perch  ?  Is  it  able  to  fly  ? 

V.  REMOVAL  OF  THE  CEREBELLUM  OF  A  PIGEON. 

Etherize  another  pigeon.  Remove  the  bone  of  the  skull  over  the 
cerebellar  region,  leaving  a  bony  bridge  in  the  middle  line.  Go  in 
from  either  side  with  a  blunt  instrument.  Sew  skin  over  wound 
and  allow  twenty-four  hours  for  recovery  from  the  shock  of  the 
operation. 

How  does  this  pigeon  compare  with  the  normal  and  with  the  one 
which  has  had  its  cerebrum  removed?  Describe  all  phenomena 
and  reactions  to  various  stimuli.  Are  there  any  disturbances  of  co- 
ordination ?  If  so,  what  are  they  ? 

VI.  HEMISECTION  OF  THE  SPINAL  CORD. 

Narcotize  a  dog  or  a  rabbit  with  morphine  and  ether.  Cut  off 
hair  of  back  in  upper  lumbar  region.  Make  a  longitudinal  incision 
through  the  skin  and  muscles  over  the  spinous  processes  of  the  first 
three  vertebra.  Cut  through  the  spinous  processes  with  bone-scis- 
sors. Clean  the  lamina?  of  muscle.  With  a  small  trephine,  care- 
fully remove  a  button  of  bone  from  the  lamina  of  one  side.  From 
this  opening  remove  the  rest  of  the  lamina  with  fine  bone-cutting 
forceps.  This  will  expose  the  cord  in  its  membranes.  Make  an 
incision  through  the  membranes  with  fine-pointed  scissors.  With  a 
fine  sharp  scalpel  cut  through  one-half  of  the  cord.  After  any  en- 
suing hemorrhage  has  been  controlled,  sew  up  the  wound  with  cat- 
gut or  silk.  Allow  the  animal  twenty-four  or  thirty-six  hours  to 
recover  from  the  shock  of  the  operation  and  then  make  observa- 
tions on  the  affected  side  compared  with  the  other  for  changes  in 
voluntary  motion  and  sensation.  Test  the  various  reflexes  also 

[52] 


NERVOUS  SYSTEM. 


and  compare  with  the  normal.  Observe  the  animal  from  day  to 
day  and  note  any  change  in  motion  or  sensation.  Try  the  muscles 
for  the  reaction  of  degeneration. 

VII.    STIMULATION    OF    THE    MOTOR    AREAS    OF    THE    DOG'S 
BRAIN.    DEMONSTRATION. 


Lightly  narcotize  a  dog 
ether.  Tie  on  dog-board, 
out  and  supported  on  a 
block  of  wood  placed  be- 
neath it.  With  the  trephine 
remove  a  button  of  bone 
from  one  parietal.  This 
opening  may  be  enlarged 
by  repeating  the  trephin- 
ing several  times.  The  re- 
mainder of  the  bone  may 
then  be  removed  piecemeal 
with  bone  forceps  and  scis- 
sors. Expose  in  this  man- 
ner the  whole  lateral  and 
dorsal  aspect  of  one  cere- 
bral hemisphere.  Identify 
the  fissures  and  motor 
points  as  shown  in  Fig.  20. 

With  fine  platinum  elec- 
trodes, having  the  two 
poles  but  slightly  sepa- 
rated, stimulate  at  the 
points  indicated  in  the 
figure.  The  current  used 
for  this  purpose  is  a  tet- 
anizing  current  from  an 
inductorium  of  medium 
strength. 


with  morphine  and  anaesthetize  with 
belly  down,  with  head  well  stretched 


FIG.  20.  —  Dog's  Brain,  Showing  Various 
Motor  Areas.  P,  Frontal  fissure,  sometimes 
termed  crucial  sulcus,  corresponding  to  the 
fissure  of  Rolando  in  man.  i,  Flexion  of  head 
on  neck  in  median  line ;  2,  flexion  of  head  on 
neck  with  rotation  towards  side  of  stimulus ; 
3,  4,  flexion  and  extension  of  anterior  limb; 
5,  6,  flexion  and  extension  of  posterior  limb ; 
7,  8,  9,  contraction  of  orbicularis  oculi,  and 
facial  muscles  in  general.  The  unshaded  part 
is  that  exposed  by  opening  the  skull.  (Dai- 
ton.) 

[53] 


LABORATORY  MANUAL  OF  PHYSIOLOGY. 


VIII.  DIVISION  OF  THE  SEMICIRCULAR  CANALS. 

A  young  pigeon  serves  best  for  this  purpose.  It  is  well  to  make 
a  dissection  on  a  dead  bird  first  in  order  to  become  familiar  with 
the  position  and  relations  of  the  canals.  Make  a  transverse  in- 
cision through  the  skin  of  the  head.  Slip  these  flaps  back  so  as  to 
expose  the  bone.  Scrape  away  the  insertions  of  the  neck  muscles. 

3 

s». 

I 


\ 


FIG.  2i.— Semicircular  Canals  (Pigeon).  Outer  plate  of  skull  and  cancellous  bone 
removed  to  expose  the  semicircular  canals  :  i,  Superior  (vertical);  2,  posterior  (verti- 
cal) ;  and  3,  anterior  (horizontal) .  The  planes  of  i,  2,  and  3  cut  each  other  at  right 
angles. 

Remove  the  outer  table  of  the  skull  behind  each  ear,  carefully  re- 
moving with  the  forceps  the  cancellous  or  spongy  bone  between  the 
two  plates  until  the  canals  are  seen  (see  Fig.  21). 

Having  made  the  preliminary  dissection  on  a  dead  bird,  repeat 
the  same  process  on  a  live  pigeon  under  the  influence  of  chloro- 
form. After  the  canals  are  exposed,  cut  through  one  or  two  of 
them,  with  strong  scissors,  making  a  careful  record  of  the  canals 
thus  injured.  Control  the  bleeding  with  suprarenal  extract. 

If  the  bird  recovers  from  the  immediate  effects  of  the  operation, 
carefully  observe  and  note  its  departure  from  the  normal  condi- 
tion of  the  pigeon  with  the  semicircular  canals  intact.  Compare 
the  behavior  of  this  pigeon  with  that  of  the  pigeon  which  had  its 
cerebellum  removed. 

[54] 


NERVOUS  SYSTEM. 

IX.  To  DETERMINE  THE  NUMBER  OF  IMPULSES  DISCHARGED  BY 
A  NERVE  CELL  IN  A  GIVEN  UNIT  OF  TIME. 

In  an  etherized  rabbit,  adopting  the  same  methods  as  were  em- 
ployed in  the  experiment  on  hemisection  of  the  cord,  expose  the 
cord  in  the  middle  lumbar  region.  Insert  needle  electrodes  in  the 
gastrocnemius  muscle  of  one  side.  Connect  these  with  the  capil- 
lary electrometer.  Insert  fine  needle  electrodes  through  the 
cord.  Stimulate  the  cord  with  medium  strong  single  induction 
shocks  at  intervals  of  one  second.  Is  there  any  response  of  the 
gastrocnemius  muscle  ?  If  so,  what  is  the  nature  of  this  response  ? 
Increase  the  frequency  of  stimulation  of  the  cord  and  note  results. 
Stimulate  the  cord  ten  times  per  second.  Does  the  muscle  go  into 
tetanus  ?  Does  the  muscular  contraction  continue  after  the  stimu- 
lation of  the  cord  has  been  stopped  ?  What  changes  occur  in  the 
capillary  electrometer  ? 

Place  the  tuning-fork  interrupter  in  circuit  with  the  primary  coil 
of  the  inductorium.  Stimulate  the  cord  again.  Does  the  muscle 
go  into  tetanus?  What  is  the  frequency  of  vibration  of  the  me- 
niscus of  the  capillary  electrometer  ? 

Listen  with  a  stethoscope  to  the  muscle  during  contraction.  Is 
the  tone  of  the  tuning-fork  reproduced  in  the  muscle?  If  not,  is 
there  any  sound  heard  in  connection  with  the  contraction  ? 

Compare  these  results  with  those  obtained  when  the  sciatic  nerve 
was  stimulated  in  the  experiment  on  muscle  tone.  What  is  the 
rate  of  discharge  from  the  nerve  cells  in  the  cord  ? 


ESS] 


CHAPTER  IV. 

BLOOD. 

THE  blood  may  be  looked  upon  as  the  common  carrier  of  the 
body.  It  serves  to  carry  food  stuffs  to  the  tissues  from  the  aliment- 
ary canal  where  they  have  been  absorbed  and  O  and  CO,  be- 
tween the  lungs  and  the  tissues.  It  also  carries  away  from  the  tis- 
sues waste  products,  resulting  from  their  metabolism,  to  the  organs 
of  excretion.  It  acts  as  a  medium  of  exchange  between  the  tissues 
themselves,  carrying  products  of  glandular  activity  from  one  group 
of  cells  to  another,  as  in  the  internal  secretions.  It  is  a  prime  factor 
in  the  regulation  of  body  temperature.  It  is  finally,  in  part,  the  re- 
ceptacle for  and,  in  part,  the  seat  of  the  formation  of  protective 
substances  which  are  manufactured  by  the  body  as  a  result  of  the 
introduction  of  toxins  from  without. 

In  structure,  the  blood  consists  of  two  main  elements,  a  liquid 
portion  or  plasma,  and  a  cellular  portion,  corpuscles.  The  latter 
are  divisible  into  two  classes,  the  colored  corpuscles  or  erythro- 
cytes  and  the  colorless  corpuscles  or  leucocytes.  They  are  also 
known  respectively  as  the  red  and  white  corpuscles.  Their  num- 
bers, varieties,  and  properties  will  be  considered  later. 

I.  COAGULATION  OF  THE  BLOOD. 

Narcotize  and  etherize  a  dog  or  rabbit.  The  former  will  furnish 
more  blood.  Expose  both  carotid  arteries.  Introduce  a  cannula 
into  each  carotid,  securing  the  arteries  on  the  side  near  the  heart 
with  artery  clamps. 

i.  Prepare  a  series  of  test  tubes,  as  follows:  (a)  Clean  empty 
tube  for  receiving  a  sample  of  fresh  shed  blood;  (b)  tube  half  full 

[56] 


BLOOD. 

of  distilled  water;  (c)  tube  half  full  of  o.8-per-cent  NaCl  solution; 
(d)  tube  half  full  of  saturated  NaCl  solution;  (e)  larger  tube 
quarter  filled  with  a  saturated  solution  of  MgSO4. 

Open  clamp  on  one  carotid  and  complete  the  filling  of  the  test 
tubes  with  blood.  Set  tube  (e)  to  one  side  for  use  later.  Observe 
what  happens  in  tube  (a),  which  contains  undiluted  fresh  blood. 
How  long  before  the  blood  in  tube  (a)  is  completely  solidified? 
Invert  test  tube.  The  blood  does  not  run  out,  but  adheres  to  the 
sides  of  the  tube  as  a  jelly-like  mass  of  the  same  volume  and  color 
throughout  as  when  first  shed.  Later,  the  mass  shrinks,  the  sur- 
face becoming  cup-shaped  and,  as  the  shrinking  continues,  more 
and  more  of  a  clear  straw-colored  fluid  collects  upon  it.  This 
is  the  serum  which  is  not  subject  to  further  coagulation,  except 
that  caused  by  high  temperatures  in  any  albuminous  fluid.  The 
solid  mass  remaining  finally  floats  in  the  serum  as  this  accumu- 
lates; it  consists  of  a  stringy  substance,  fibrin,  and  blood  corpuscles 
entangled  in  its  network-like  meshes. 

If  a  little  fresh  blood  be  allowed  to  drop  on  a  glass  slide;  and  is 
then  covered  with  a  cover  slip  and  placed  in  a  moist  chamber  to 
prevent  drying,  after  fifteen  to  twenty  minutes  the  fibrin  fibrils  may 
be  seen  with  a  low  power  of  the  microscope. 

Has  the  distilled  water  of  tube  (b)  any  effect  in  hastening  or  de- 
laying the  coagulation  of  the  blood  shed  into  it  ? 

Has  the  o.8-per-cent  NaCl  solution  of  tube  (c)  any  effect  in  hast- 
ening or  delaying  the  coagulation? 

What  is  the  effect  of  the  saturated  salt  solution  of  tube  (d)  ? 

2.  Compare  the  color  of  the  fresh  undiluted  blood  with  the  sat- 
urated salt-solution  dilution  and  the  distilled-water  dilution.    Com- 
pare the  different  tubes  in  transmitted  and  reflected  light.     To 
what  is  the  opacity  of  the  fresh  blood  due  ?    To  what  is  the  trans- 
parency of  the  water-diluted  blood  due? 

3.  Place  a  specimen  from  each  tube  under  the  microscope  and 
compare  the  appearances  of  the  red  corpuscles.     With  distilled 
water  and  some  other  reagents  the  red  blood  corpuscles  lose  their 
pigment  (haemoglobin)  which  goes  into  solution  in  the  diluted 

[57] 


LABORATORY  MANUAL  OF  PHYSIOLOGY. 

plasma,  giving  it  a  transparent  color.  This  process  is  known  as 
taking  and  the  blood  is  said  to  be  laked.  The  corpuscles  will  ap- 
pear faintly  outlined  as  ghost  or  shadow  corpuscles. 

4.  The  magnesium  sulphate  of  tube  (e)  prevents  coagulation. 
Let  the  mixture  stand  in  a  cool  place  until  the  corpuscles  have  set- 
tled to  the  bottom  of   the  tube.     The  supernatant  liquid,  the 
"salted  plasma,"  may  then  be  pipetted  or  siphoned  off. 

Divide  this  salted  plasma  into  four  portions.  To  each  portion 
add  eight  times  its  volume  of  water.  To  portion  i  add  a  few  drops 
of  a  half-per-cent  solution  of  ammonium  oxalate.  To  portion  2 
add  a  little  of  the  clot  from  tube  (a).  Portion  3,  place  in  a  water 
bath  heated  to  38°  C.  Place  portion  4  on  a  water  bath  heated  to 
60°  C.  and  add  a  few  drops  of  ammonium  oxalate. 

Observe  the  presence  or  absence  of  the  phenomena  of  coagulation 
in  the  portions  of  salted  plasma  treated  as  above.  Are  calcium 
salts  necessary  to  coagulation  ?  What  is  the  effect  of  tempera- 
ture on  coagulation  ?  Why  does  coagulation  take  place  in  por- 
tion 2? 

To  tubes  i  and  4  add  a  few  drops  of  calcium  chlorid.  What  is 
the  effect  as  far  as  coagulation  is  concerned  ? 

There  are  various  theories  to  explain  the  coagulation  of  the 
blood.  The  known  facts  are  as  follows:  Clotting  is  produced 
through  the  formation  of  a  coagulated  substance,  fibrin;  for  the 
formation  of  fibrin  three  things  are  necessary:  a  globulin,  fibrin- 
ogen,  calcium  salts,  and  an  enzyme,  fibrin  ferment  or  thrombin. 

Fibrinogen  and  soluble  calcium  salts  are  normally  present  in  the 
blood  plasma.  Thrombin  is  formed  at  the  time  of  coagulation. 
The  mooted  question  is  the  origin  of  the  thrombin.  The  thrombin 
is  a  nucleo-proteid  which  seems  to  be  formed  through  cell  disinte- 
gration and  especially  through  the  breaking  down  of  leucocytes. 

5.  Defibrination  of  Blood. — To  defibrinate  blood,  collect  it 
from  a  bleeding  artery,  in  a  shallow  vessel.    As  the  blood  is  shed, 
whip  or  beat  it,  vigorously,  with  a  glass  rod  or  a  bundle  of  twigs. 
The  fibrin,  as  it  is  formed,  separates  from  the  blood  and  adheres 
to  the  whip  as  a  sticky,  stringy,  almost  colorless  mass.    The  blood 

[58] 


BLOOD. 

so  treated  is  then  filtered  through  a  fine-mesh  cloth.    Defibrinated 
blood  will  not  clot  spontaneously. 

II.  THE  NUMBER  OF  RED  AND  WHITE  BLOOD  CORPUSCLES. 

1.  Counting  the   Erythrocytes   or   Red   Corpuscles. — The 

Thoma-Zeiss  haemocytometer  is  used  for  this  purpose.    This  con- 


FlG.  22. — Thoma-Zeiss  Haemocyto meter  Counting-chamber,  s,  Glass  slide,  upon 
which  is  mounted  a  covered  disc  m,  accurately  ruled  to  present  one  square  millimetre 
divided  into  400  squares.  This  is  surrounded  by  another  annular  cell,  c,  which  pro- 
jects in  height  exactly  one  tenth  of  a  millimetre  above  m. 

sists  of  a  graduated  pipette  for  accurately  diluting  a  known  quan- 
tity of  blood  with  some  fluid  having  the  same  osmotic  pressure  as 
the  blood.  One  of  the  most  satisfactory  diluents  is  physiological 
salt  solution.  For  human  blood,  this  consists  of  a  solution  of  so- 
dium chlorid,  8.5  grams  in  1000  c.c.  of  water. 

The  capillary  stem  of  the  pipette,  used  for  diluting  the  blood  for 
counting  the  red  corpuscles,  has  a  capacity  equalling  one-hun- 
dredth of  the  hollow  ball  with  which  it  joins  (see  Fig.  23).  If  the 
blood  is  drawn  up  to  the  line  marked  i  on  the  pipette  stem  and 
then  the  diluent  drawn  in  until  the  mixture  reaches  the  line  101 


FIG.  23.— Thoma-Zeiss  Haemocytometer  Pipette. 

marked  just  above  the  ball  (see  Fig.  23),  there  will  be  101  parts  of 
fluid,  of  which  the  blood  forms  i.  The  contents  of  the  stem,  how- 
ever, do  not  have  to  be  considered  after  the  dilution  is  made,  since 
they  can  be  displaced,  unmixed.  The  dilution  of  the  blood  will 
then  be  i  to  100.  As  the  blood  and  diluting  fluid  enter  the  mixing 

[59] 


LABORATORY  MANUAL  OF  PHYSIOLOGY. 

chamber,  this  should  be  constantly  rotated  between  the  fingers  so 
as  to  facilitate  the  mixture  and  avoid  error  through  coagulation. 

The  other  part  of  the  instrument  is  the  micrometer  slide  upon 
which  the  diluted  blood  is  evenly  spread  for  counting  the  corpus- 
cles. This  consists  of  a  glass  slide  (Fig.  22),  upon  which  is  mounted 
a  covered  disc,  m,  a  square  millimetre  of  which  is  subdivided  by  a 
dividing  engine  into  400  squares  of  one-twentieth  millimetre  each. 
The  micrometer,  m,  is  surrounded  by  an  annular  cell,  c,  the  sides 
of  which  project  one-tenth  millimetre  above  the  surface  of  m.  This 
cell  is  closed  by  a  thin  flat  glass  cover,  so  that  the  cubic  space  in- 
cluded between  each  small  square  of  the  micrometer  and  the  cover 
would  be  ^nnr  °f  a  cubic  millimetre. 

To  find  the  number  of  corpuscles  in  a  cubic  millimetre  of  undi- 
luted blood,  multiply  4000  by  the  dilution  and  this  by  the  total 
number  of  corpuscles  counted.  This  result  is  then  divided  by  the 
number  of  small  squares  counted.  If  the  blood  has  been  drawn 
only  to  the  0.5  mark  in  the  diluting  pipette,  the  blood  dilution  is 
i  to  200  and  this  number  must  be  substituted  for  the  factor  100  in 
the  formula  given  above.  With  normal  blood,  the  higher  dilution 
is  advisable. 

Procedure. — Thoroughly  cleanse  the  tip  of  the  finger  or,  prefer- 
ably, the  lobe  of  the  ear,  with  soap  and  water.  Wipe  off  with  a 
cloth  wet  with  alcohol.  Dry  thoroughly.  With  a  sterilized  needle, 
or  a  sharp  pen  with  one  nib  broken  off,  make  a  quick  stab  of  the 
ear  or  the  finger.  Wipe  off  the  first  drop  of  blood.  Blood  should 
ooze  freely  from  the  puncture  without  pressure.  Insert  the  point 
of  the  pipette  well  into  the  blood  drop  and  carefully  draw  in  blood 
to  the  0.5  mark  on  the  stem  of  the  pipette.  With  a  cotton  cloth 
wipe  off  all  blood  adhering  to  the  outside  of  the  pipette.  Dip  the 
end  of  the  pipette  into  the  diluting  fluid  and  draw  this  in  through 
the  stem  and  into  the  ball  until  the  101  mark  is  reached.  The  pi- 
pette should  be  gently  rotated  while  the  filling  is  going  on,  in  order 
that  the  mixture  of  the  blood  and  diluting  fluid  may  be  assured 
through  the  movements  of  the  glass  bead  in  the  ball.  Close  both 
ends  of  the  pipette  with  thumb  and  forefinger  and  shake  well. 

[60] 


SLOOD. 

This  is  to  obtain  a  uniform  distribution  of  the  corpuscles  through- 
out the  mixture. 

To  Fill  the  Counting  Cell. — Blow  out  three  or  four  drops  of  the 
diluted  blood  from  the  pipette.  Now  allow  a  small  drop  to  flow 
upon  the  disc  of  the  counting  chamber.  Cover  quickly,  pressing 
the  cover  gently  down  until  Newton's  rings  are  seen.  These  are 
the  spectrum  colors  due  to  refraction  between  the  two  layers  of 
glass.  They  do  not  appear  if  there  is  any  fluid  or  dirt  between  the 
cover  and  the  cell.  If  any  fluid  runs  over  into  the  moat  between 
the  cell  and  the  micrometer,  the  slide  will  have  to  be  cleaned  and 
another  drop  of  the  diluted  blood  taken.  Repeat  until  a  satis- 
factory specimen  for  counting  is  obtained. 

Allow  several  minutes  for  the  corpuscles  to  sink  to  the  bottom  of 
the  cell  upon  the  ruled  squares.  It  is  obvious  that  the  counting  cell 
must  ke  kept  in  the  horizontal  position.  Place  this  upon  the  stage 
of  a  microscope  and  count  the  corpuscles  in  all  the  squares.  For 
convenience  of  counting,  the  micrometer  is  divided  into  sixteen 
large  squares  by  double  lines,  and  these,  in  their  turn,  are  sub- 
divided into  the  small  squares  already  mentioned.  Count  several 
specimens,  in  this  way,  and  take  the  average.  Compare  the 
blood  of  various  students. 

To  Clean  the  Cell  and  Pipette.— The  cell  should  be  carefully  rinsed 
with  distilled  water  and  dried  with  a  soft  cloth  or  absorbent  cot- 
ton. The  cover  should  be  treated  in  the  same  way.  Neither  alco- 
hol nor  ether  should  be  used  since  they  will  coagulate  the  albumin 
of  the  blood.  In  cleaning  the  pipette  first  blow  out  any  blood 
mixture  remaining.  Fill  with  distilled  water  several  times.  If  all 
traces  of  blood  are  not  removed  in  this  way,  rinse  with  an  aqueous 
solution  of  hydrogen  peroxide  and  again  wash  out  with  distilled 
water.  Now  draw  alcohol  through  by  suction  and  follow  this  with 
ether,  drawing  through  a  stream  of  air  until  the  pipette  is 
thoroughly  dry.  This  is  manifest  when  the  glass  bead  enclosed  in 
the  bulb  of  the  pipette  no  longer  adheres  to  the  sides. 

Counting  the  White  Corpuscles. — Since  there  is  a  much  smaller 
number  of  leucocytes  than  of  red  corpuscles,  the  dilution  required 

[61] 


LABORATORY  MANUAL  OF  PHYSIOLOGY. 


is  much  less.  A  special  pipette  is  employed,  with  which  a  dilution 
of  i  to  i o  or  i  to  20  may  be  obtained.  The  diluting  fluid  employed 
is  usually  a  0.2  of  one  per  cent  acetic  acid.  This  accentuates  the 


f 

FIG.  24. — Human  Blood-corpuscles,  a,  Red  blood-corpuscles  for  comparison;  £, 
small  hyaline  cell  or  small  lymphocyte ;  c,  large  hyaline  cell  or  large  lymphocyte ;  </, 
fine  granular  oxyphile  ;  e,  coarse  granular  oxyphile  or  eosinophile ;  /,  basophile.  (F. 
C.  Busch.) 

nuclei  of  the  white  cells  and  decolorizes  the  reds.    Aside  from  this, 
the  technique  of  counting  is  the  same  as  that  for  the  reds. 

II.  CHANGES  PRODUCED  IN  THE  CORPUSCLES  THROUGH  VARI- 
ATIONS OF  OSMOTIC  PRESSURE. 

Dilute  equal  volumes  of  defibrinated  blood  with  (a)  distilled 
water;  (b)  o.8-per-cent  sodium- chlorid  solution;  (c)  5-per-cent 
sodium-chlorid  solution. 

Note  the  color  and  opacity  of  (b)  and  (c)  as  compared  with  (a) . 
Place  samples  of  the  three  preparations  under  the  microscope. 
Note  the  changes  of  form  and  color  of  the  corpuscles :  (b)  is  an  iso- 
tonic  solution  for  the  blood,  (c)  is  hyperisotonic,  and  (a)  is  hypo- 
isotonic. 


BLOOD. 

With  fresh  preparations  on  a  slide  and  covered  with  a  cover 
glass,  try  the  effects  of  the  following  reagents:  acetic  acid,  chloro- 
form, ether. 

Place  another  specimen  of  denbrinated  blood  in  a  freezing  mixt- 
ure. 

III.  MICROSCOPIC  EXAMINATION  OF  THE  BLOOD. 

Appliances. — Microscope,  with  a  substage  condenser,  a  high  dry 
and  an  oil-immersion  lens.  A  stage  micrometer.  An  eyepiece  mi- 
crometer. Slides  and  cover  slips.  Staining  reagents,  consisting  of 
methylene  blue  (aqueous  solution),  eosin  (sat.  aqueous  solution), 
Wright's  stain. 

The  slides  and  cover  slips  used  must  be  scrupulously  clean. 
With  new  slides,  washing  with  soap  and  warm  water,  and  then 
rinsing  in  distilled  water,  are  generally  sufficient.  The  glasses 
should  then  be  wiped  dry  with  a  clean  soft  linen  or  cotton  cloth. 
Handling  the  surfaces  of  the  slides  or  cover  slips  with  the  fingers 
should  be  avoided,  since  the  oil  from  the  skin  deposited  in  this  way 
is  hard  to  remove  and  prevents  an  even  spreading  of  the  blood- 
film.  Before  using  the  slides  or  cover  slips,  they  should  be  gently 
warmed  for  a  moment  in  the  flame  of  the  alcohol  lamp  or  Bunsen 
burner  in  order  to  get  rid  of  any  water  of  condensation. 

i .  In  the  manner  described  before,  obtain  a  drop  of  blood  from 
the  finger  or  ear.  Touch  the  middle  of  a  clean  glass  slide  to  the 
drop  so  that  a  small  portion  adheres  to  the  slide.  Avoid  touching 
the  skin  of  the  ear  with  the  slide.  Gently  place  a  clean  cover  slip 
over  the  drop  and  allow  the  blood  to  spread  out  in  a  thin  film  be- 
tween the  slide  and  the  cover  glass  without  using  any  additional 
pressure. 

Examine  this  fresh  specimen  under  the  microscope  with  the  high 
dry  lens.  Note  the  color  and  form  of  the  red  cells.  Compare  them 
with  the  leucocytes  in  relative  number  and  form.  Are  the  red  cells 
nucleated?  What  is  the  appearance  of  the  red  cells  in  profile? 
Do  the  red  cells  vary  in  size  and  form  ?  If  so,  to  what  may  these 
variations  be  attributed  ? 


LABORATORY  MANUAL  OF  PHYSIOLOGY. 

2.  Instead  of  the  blood  slide,  place  the  stage  micrometer  under 
the  microscope.    With  the  help  of  this,  find  the  exact  value  of  the 
spaces  between  the  lines  of  the  ocular  micrometer.    Remove  the 
stage  micrometer  and  again  place  the  blood  specimen  under  the 
microscope.    With  the  ocular  micrometer,  measure  the  dimensions 
of  twenty-five  red  corpuscles.    Compare  the  size  of  the  white 
corpuscles  with  that  of  the  reds. 

3.  Pith  a  frog.    From  the  wound  thus  made,  secure  some  blood 
for  microscopical  examination.    How  do  the  red  blood  cells  of  the 
*rog  compare  in  size,  form,  and  nucleation  with  the  red  cells  of  man 
and  of  the  rabbit  or  dog  ?    In  frog's  blood  what  is  the  relative  size 
of  red  and  white  cell  ? 

IV.  STAINING  OF  THE  BLOOD  CELLS  AND  DIFFERENTIAL  COUNT 
OF  THE  LEUCOCYTES. 

A  dried  blood  smear  or  film  is  usually  employed  for  this  pur- 
pose. The  blood  smear  may  be  made  in  one  of  two  ways,  either 
on  a  slide  or  upon  a  cover  slip.  If  the  former  method  is  employed, 
the  smear  is  allowed  to  dry  on  the  slide,  is  stained,  and  examined, 
without  the  use  of  a  cover  glass.  If  the  latter  method  is  used,  the 
smear  is  dried,  fixed,  and  stained  upon  the  cover  slip  and  this  is 
then  inverted,  smear  side  down,  upon  a  slide  over  a  drop  of  balsam 
or  a  shallow  air  cell. 

1.  The  Smear  on  the  Slide  Direct. — Take  two  slides.    Touch 
the  edge  of  one  to  the  drop  of  blood.     With  this  slide  forming  an 
angle  of  about  25°  with  the  other,  quickly  and  firmly  apply  its 
blood-stained  edge  to  the  other  slide  and  sweep  it  over  the  surface. 

2.  The   Cover-Glass    Smear. — For  this  purpose,  two  cover 
slips  are  used.    One  of  the  cover  slips  is  carefully  applied  to  the 
drop  of  blood,  so  that  a  drop  adheres  to  the  centre  of  the  slip.    This 
slip  is  then  applied  to  the  other  and  the  blood  allowed  to  spread  in 
a  thin  film  between  the  two.    After  this  has  occurred,  the  two  slips 
are  carefully  drawn  apart  and  the  smears  allowed  to  dry  in  the  air. 

3.  Staining   the   Films. — The    simplest  and   best   available 
blood-staining  reagent  at   the  present  time  is  that  devised  by 


BLOOD. 

Wright.  This  is  a  modification  of  the  method  used  by  Jenner. 
The  method  is  based  upon  the  fixing  and  solvent  powers  of  me- 
thylic  alcohol.  The  essential  pigments  of  the  stain  are  polychrome 
methylene  blue  and  Griibler's  yellow  eosin. 

(a)  Cover  the  blood  film  on  the  slide  or  cover  glass  with  as  much 
of  the  stain  as  it  will  hold.    Allow  this  to  remain  undisturbed  for 
about  one  minute.    By  this  time  the  film  is  fixed  upon  the  slide. 

(b)  Now  add  water,  drop  by  drop,  until  the  surface  of  the  stain 
assumes  a  greenish  metallic  tinge.    Allow  the  stain  to  remain  on  the 
cover  slip  for  two  minutes  longer. 

(c)  Wash  in  tap  water  for  two  minutes  or  until  the  smear  has 
acquired  a  yellowish-pink  hue.    The  water  serves  to  differentiate 
the  stain  and  wash  out  the  excess  of  blue. 

(d)  Dry  the  specimen  carefully  with  filter  paper  and  mount  in 
Canada  balsam. 

4.  Iodine  Reaction. — The  reaction  which  iodine  gives  with 
the  finely  granular  oxyphile  cells  or  polynuclear  neutrophiles,  is 
known  as  iodophilia.  The  following  solution  is  employed: 

Iodine i  gm. 

Potassium  iodide 3     " 

Water 100  c.c. 

Gum  Arabic 50  gm. 

Place  a  drop  of  this  mixture  upon  a  slide.  Take  a  fresh  blood 
film  on  a  cover  slip  and  press  it,  film  down,  upon  this  mixture. 
Squeeze  out  from  under  the  cover  slip  the  excess  of  the  mixture, 
so  that  the  remaining  film  is  sufficiently  thin  to  avoid  obscuring  the 
corpuscles  through  too  deep  a  color  of  the  mounting  medium. 

In  normal  blood,  all  the  cells  will  be  tinged  a  bright  yellow: 
the  reds,  uniformly;  the  whites,  with  a  more  refractile  nucleus. 
In  certain  pathological  conditions,  with  this  treatment  of  the  blood, 
reddish-brown  granules  appear  in  the  cytoplasm  of  the  polynuclear 
neutrophiles  as  well  as  granular  masses  of  a  similar  tinge  outside  of 
the  cells. 


[65] 


LABORATORY  MANUAL  OF  PHYSIOLOGY. 

V.  CLASSIFICATION  OF  THE  LEUCOCYTES. 

There  is,  at  present,  no  perfectly  satisfactory  classification  of 
the  white  cells.  One  of  the  most  logical  is  that  of  Kanthack  and 
Hardy  (see  Fig.  24).  This  is  a  classification  according  to  reaction 
to  staining  reagents  and  to  presence  or  absence  of  granulation  and 
is  as  follows: 

A.  Oxyphile  (staining  with  acid  dyes). 

1 .  Finely  granular. 

2.  Coarsely  granular. 

B.  Basophile  (staining  with  basic  dyes). 

i.  Finely  granular. 

C.  Hyaline. 

1.  Small. 

2.  Large. 

The  more  usual  classification,  however,  is  as  follows:  (a)  poly- 
morphonuclear  neutrophiles,  (b)  eosinophiles,  (c)  mast  cells,  (d) 
large  mononuclear  cells,  (e)  lymphocytes  (large  and  small). 

Of  these,  (a)  to  (d}  inclusive  originate  in  the  bone  marrow. 
Group  (e)  comes  from  adenoid  tissue. 

1.  Differential  Count. — Study  one  of  the  best  stained  speci- 
mens of  human  blood  until  you  are  familiar  with  the  different 
forms  of  leucocytes  enumerated  above.  Now  go  over  the  speci- 
men carefully  and  systematically,  using  a  mechanical  stage  so  as 
not  to  go  over  the  same  field  twice,  and  keep  count  of  the  number 
of  individuals  of  the  different  varieties.  Count  five  hundred  cells 
in  all  and  estimate  the  percentage  of  each  form.  At  the  same 
time  keep  careful  watch  for  any  abnormalities  of  the  reds. 

Stain  a  specimen  of  frog's  blood  and  compare  the  varieties  of 
white  cells  with  those  found  in  human  blood. 

Stain  smears  of  human  blood  with  eosin  and  methylene  blue, 
after  fixation  for  two  hours  in  a  mixture  of  alcohol  and  ether,  equal 
parts. 

[66] 


BLOOD. 

Normal  Percentage  of  Each  Variety  (Cabot). 
Small  lymphocytes   ............  .  .............    20-30  per  cent. 

Large  "  ...........................  4-8 

Polymorphonuclear  neutrophiles  ................  62-70          " 

Eosinophiles  .................................   5-4 

"  Mast  cells" 


Description  of  the  Different  Varieties.  —  The  variety  which  is  most 
numerous,  as  may  be  seen  from  the  above  table,  is  the  so-called 
polynuclear  or  polymorphonuclear  neutrophile.  With  Wright's 
stain,  the  nucleus  of  this  corpuscle  takes  an  intense  navy-blue  color 
and  is  sharply  defined.  The  nucleus  is  irregular  in  outline  and 
may  assume  a  great  variety  of  forms.  There  may  be  two  or  more 
nuclear  masses  united  by  finer  bands  of  nuclear  substance.  The 
cytoplasm  contains  fine  granulations  which  take,  with  Wright's 
stain,  several  different  shades  of  pink.  Where  the  simple  eosin 
and  methylene-blue  staining  is  employed,  these  granules  take  a 
faint  pink  tinge. 

The  lymphocytes,  which,  in  point  of  number,  come  next  to  the 
•polynuclear  neutrophile,  vary  considerably  in  size,  from  that  of  a 
red  blood  corpuscle  to  several  times  the  size  of  a  red.  The  nucleus 
is  large,  generally  round,  but  may  be  oval  or  bean-shaped.  There 
is  but  a  narrow  rim  of  cytoplasm  around  the  nucleus.  The  cyto- 
plasm, with  Wright's  stain,  takes  a  robin  's-egg-blue  tint  and  the 
nucleus  stains  a  deep  purple  or  purple-blue. 

Granules  are,  as  a  rule,  absent.  Forms  are  seen,  however,  in 
which  the  cytoplasm  contains  from  a  few  to  a  large  number  of 
pinkish  but  more  generally  blue  granules. 

The  eosinophile  is  a  large  cell,  resembling,  in  structure  and 
size,  the  polynuclear  neutrophile.  With  Wright's  stain,  the  nuclei 
stain  light  blue  or  lilac,  with  an  ill-defined  intranuclear  network. 
The  large  spherical  or  oval  granules  take  a  brilliant  red  eosin 
stain.  The  cytoplasm  around  the  granules  either  takes  no  stain 
whatever  or  a  pale  blue  tinge.  This  type  of  leucocyte  occurs  nor- 
mally in  very  small  numbers,  and  a  number  of  fields  may  be  gone 
over  with  the  microscope  before  an  eosinophile  is  found. 

[67] 


LABORATORY  MANUAL  OF  PHYSIOLOGY. 

The  mast  cell  is  not  commonly  found  in  normal  blood  and  you 
will  probably  not  see  it  among  the  comparatively  small  number  of 
white  cells  that  you  are  required  to  count.  The  cell  is  about  twice 
the  diameter  of  the  red  cell,  has  a  polymorphous  nucleus  of  vague 
outlines,  and  a  cytoplasm  containing  numerous  very  large  granules 
taking  a  dark  blue  or  blue-black  stain. 

VI.  ESTIMATION  OP  HEMOGLOBIN. 

A  number  of  instruments  have  been  devised  for  estimating  the 
haemoglobin  of  the  blood.  In  most  of  these,  for  convenience  of 
comparison,  a  scale  of  100  is  used,  the  100  mark  corresponding  to 
a  haemoglobin  content  of  13.8  grams  of  haemoglobin  in  100  c.c.  of 
blood.  The  instruments  as  a  rule  depend  on  a  color  comparison 
between  the  shed  blood,  with  or  without  dilution,  and  a  fixed  scale 
of  color  to  correspond  to  various  dilutions.  There  is  a  certain  de- 
gree of  unavoidable  error  in  the  employment  of  any  color  test, 
which  at  times  may  be  very  high.  A  method  which  avoids  the  er- 
rors of  the  color  comparisons  is  the  estimation  of  the  haemoglobin 
from  the  specific  gravity. 

The  simplest,  least  expensive,  and  most  practical  scheme  of 
color  test  yet  devised  is  that  of  Talqvist. 

1.  Talqvist's  Hsemoglobinometer. — This  consists  of  a  pa- 
per scale  of  color  shades  varying  from  10  to  100  per  cent  haemo- 
globin and  contained  in  a  book  of  filter  paper  which  is  used  for 
absorbing  the  specimens  of  blood  whose  percentage  of  haemoglobin 
is  to  be  estimated.    The  blood  stain,  undiluted,  is  compared  with 
the  haemoglobin  scale  by  reflected  daylight  until  a  shade  is  found 
to  correspond  to  the  tinge  of  the  blood  examined.    For  approximate 
clinical  results  this  method  is  very  satisfactory. 

2.  Dare's  Haemoglobinometer. — As  in  the  method  of  Tal- 
qvist, undiluted  blood  is  used.    This  is  drawn  by  capillarity  be- 
tween two  plates  of  glass,  one  of  which  is  transparent,  the  other  be- 
ing translucent  for  diffusing  the  light  used  for  illumination. 

The  color  comparison  is  made  with  that  of  a  glass  disc  which  is 
revolved  by  means  of  a  thumb  screw  so  as  to  bring  successive  tints 

[68] 


BLOOD. 

into  relation  with  the  blood  specimen  being  examined.  Trans- 
mitted candle  light  is  used  for  illumination.  This  instrument  is 
more  accurate  than  the  Talqvist  device,  but  is  much  less  convenient 
and  more  expensive. 

3.  Haemometer  of  v.  Fleischl. — In  this  instrument  the 
amount  of  haemoglobin  in  a  specimen  of  blood  is  estimated  by 
comparing  a  stratum  of  diluted  blood  with  a  standard  glass  wedge 


FIG.  25.— V.  Fleischl's  Haemometer  (modified  by  Miescher). 

of  uniform  tint  spectroscopically  similar  to  that  of  the  blood.  This 
instrument  has  been  recently  modified  and  made  more  accurate 
by  Miescher. 

This  modification  of  the  instrument  consists  of  a  stand  with  a 
metal  plate  having  a  circular  opening  and  a  plaster  mirror  below 
(see  Fig.  25),  which  serves  to  reflect  the  light  through  the  colored 
wedge  and  diluted  blood.  Beneath  the  metal  plate  is  a  metal 
frame  carrying  the  colored  wedge  alongside  of  which  is  a  scale  in- 
dicating the  different  percentages  of  haemoglobin  corresponding 
to  the  varying  thicknesses  of  the  wedge.  This  framework  is 


LABORATORY  MANUAL  OF  PHYSIOLOGY. 

moved  by  the  wheel  (T),  which  fits  into  a  rack  on  its  lower  surface. 
The  scale  may  be  read  through  a  small  opening  (m)  in  the  plate. 
Into  the  large  circular  opening  of  the  plate,  fits  a  cylindrical  metal 
cell  (M)  with  a  glass  bottom  and  divided  by  a  thin  metal  partition 
into  two  equal  parts.  One  of  these  halves  lies  over  the  wedge  and 
is  filled  with  distilled  water.  The  other  contains  the  solution  of 
blood  to  be  tested.  The  apparatus  is  usually  supplied  with  three 
cells  (M,  Mf,  M").  Of  these,  the  first  two  are  used  in  estimating 
the  haemoglobin  according  to  Miescher's  modification  of  v. 
FleischPs  original  method.  These  cells  are  furnished  with  a  glass 
cover  (D),  having  a  groove  which  fits  on  the  partition  of  the  cell. 
Over  this  cover  is  placed  a  diaphragm  (Bl),  with  a  longitudinal 
slit,  which  permits  the  central  part,  only,  of  each  side  of  the  cell  to 
be  seen.  The  third  cell  (M")  is  for  use  with  the  original  method. 

Procedure. — Blood  from  the  wround  is  sucked  up  into  the  grad- 
uated pipette  (Mel)  until  it  reaches  the  mark  |  or  §  or  y.  A  one- 
per-cent  solution  of  sodium  carbonate  is  then  sucked  in  until  the 
upper  mark  is  reached.  The  pipette  is  then  well  shaken  in  order 
to  mix  the  blood  thoroughly.  One-half  of  the  two  cells  (M,  M'), 
which  are  respectively  12  and  15  mm.  high,  are  then  filled  with  the 
mixture,  the  other  half  being  filled  with  water.  The  cells  should 
be  completely  filled.  The  cover  glasses  and  diaphragm  are  then 
applied  and  the  cells  are  ready  for  examination.  Artificial  light  is 
employed.  One  of  the  cells  is  placed  on  the  plate  and  the  wheel 
(T)  turned  until  the  colors  of  the  two  halves  exactly  correspond. 
The  result  is  then  read  off  through  the  scale  opening  (m).  This 
should  be  repeated  several  times  with  each  of  the  cells  and  the 
average  of  the  readings  taken.  The  result  obtained  with  the  12 
mm.  cell  is  to  be  multiplied  by  f  to  bring  it  up  to  that  of  the  larger. 

Suppose  the  result  of  several  readings  to  be  as  follows: 

With  the  large  cell  (15  mm.) 54-oo 

With  the  small  cell  (12  mm.) 42.00 

If  the  readings  with  the  large  cell  are  exactly  correct,  the  reading 
with  the  smaller  one  should  be  43.2,  since  54  Xf  =  43.2.  Or,  if 


BLOOD. 

the  reading  with  the  small  cell  is  correct,  the  reading  with  the  large 
one  should  be  52.5,  since  42  Xf—  52.5.  The  mean  of  the  two 
readings  is  taken  as  approximately  correct. 

Each  instrument  is  supplied  with  a  corrected  scale  of  haemo- 
globin values.  Comparing  the  figure,  obtained  above,  with  the 
scale,  it  is  found  to  correspond  to  a  solution  containing  400  milli- 
grams of  haemoglobin  in  1000  c.c.  of  solution.  The  dilution  which 
was  employed  was  i  to  200,  i  to  300,  or  i  to  400,  according  as  to 
whether  the  pipette  was  filled  to  the  mark  ^,  f ,  or  y.  To  find  the 
actual  amount  of  haemoglobin  in  a  given  volume  of  blood,  the  re- 
sult obtained  would  have  to  be  mutliplied  by  200,  300,  or  400.  In 
the  example  taken  above  with  a  dilution  of  i  to  200  there  would  be 
8  grams  of  haemoglobin  in  100  c.c.  of  blood. 

4.  Estimation  of  Specific  Gravity. — It  was  first  demon- 
strated by  Hammerschlag  that  the  specific  gravity  of  blood  bore  a 
sufficient  relation  to  the  haemoglobin  content  to  make  it  of  value  in 
estimating  the  same.  Variations  in  haemoglobin  ordinarily  cor- 
respond quite  closely  to  variations  in  specific  gravity.  With  this 
in  view  Hammerschlag  devised  the  following  table  showing  the  re- 
lation between  specific-gravity  changes  and  variations  in  haemo- 
globin percentage: 

Specific  Gravity.  Per  cent  Haemoglobin 

.033-1 .035 25-30 

.035-1 .038 30-35 

.038-1.040 35-40 

.040-1 .045 40-45 

.045-1 .048 45-55 

.048-1.050 55-65 

.050-1 .053 65-70 

-053-!  -055 7°-75 

-OSS"1  -°57 75-85 

.057-1.060 85-95 

The  most  convenient  method  for  obtaining  the  specific  gravity 
is  through  the  use  of  two  fluids  with  which  the  blood  will  not  mix, 
one  of  a  high  density  and  the  other  of  a  lower  density,  such  as 
chloroform  and  benzol. 

[71] 


LABORATORY  MANUAL  OF  PHYSIOLOGY. 

The  necessary  apparatus  consists  of  a  urinometer  jar,  urino- 
meter,  a  pipette  of  small  calibre,  glass  rod,  fine  steel  pens,  bottle  of 
chloroform,  benzol,  and  a  mixture  of  the  two.  The  mixture 
should  be  approximately  that  of  the  estimated  specific  gravity  of 
the  blood  to  be  tested. 

A  drop  of  blood  is  sucked  up  into  the  pipette.  This  is  gently 
blown  out  below  the  surface  of  the  mixture,  care  being  taken  to 
leave  some  blood  in  the  pipette.  It  is  well  to  have  several  drops  in 
the  mixture.  If  the  specific  gravity  of  the  blood  and  that  of  the  mixt- 
ure is  the  same,  the  drop  will  float  indifferently  wherever  placed. 
If  the  specific  gravity  of  the  mixture  is  greater  than  that  of  the  blood 
dropj  the  latter  will  rise.  If  the  mixture  is  lighter  than  the  drop, 
the  latter  will  sink.  The  density  of  the  mixture  may  be  increased 
by  adding  chloroform  and  decreased  by  adding  benzol.  The 
mixing  of  the  two  fluids  is  accomplished  by  careful  stirring  with  a 
glass  rod.  When  the  density  of  the  blood  and  that  of  the  con- 
taining mixture  is  the  same  the  specific  gravity  is  taken  by  means 
of  the  urinometer. 

The  same  chloroform-benzol  mixture  may  be  used  repeatedly, 
if  filtered  after  each  test.  Scrupulous  cleanliness  must  be  observed. 
Otherwise,  particles  of  dust  might  adhere  to  the  blood  drop  and 
thus  cause  an  error.  Care  should  also  be  taken  to  avoid  the  ad- 
mixture of  air  with  the  drop  of  blood.  The  reading  should,  like- 
wise, be  taken  as  soon  as  possible,  to  avoid  error  through  vapor- 
ization and  through  changes  in  the  blood  drop.  If  the  hydrometer 
jar  is  not  perfectly  clean,  the  globule  of  blood  is  liable  to  adhere  to 
the  sides. 

The  advantages  of  this  method  of  haemoglobin  determination  are 
obvious.  There  is  no  delicate  color  comparison  to  be  made.  If 
proper  precautions  are  taken,  the  experimental  error  is  very  small 
as  compared  with  the  color  methods.  The  apparatus  is  simple  and 
inexpensive  and  the  technique  is  not  difficult. 

Estimate  the  haemoglobin  of  a  fellow-student  by  the  several 
methods  given  above  and  compare  results. 

[72] 


BLOOD. 

VII.    HAEMOGLOBIN   AND   ITS   DERIVATIVES. 

1.  Haemoglobin    Crystals. — Shake    up    some    defibrinated 
blood  with  CO2  gas;  add  ether,  slowly,  until  the  blood  has  become 
laked.    Set  in  the  cold  for  several  days.    Part  of  the  haemoglobin 
will  have  crystallized  out  and  may  be  removed  with  a  pipette  and 
examined  under  the  microscope. 

Haemoglobin  of  different  animals  crystallizes  with  varying  facil- 
ity. The  haemoglobin  of  man  and  of  the  herbivorous  animals  is 
very  soluble  and  crystallizes  with  great  difficulty.  That  of  the  rat 
and  guinea-pig  is  much  less  soluble  and  therefore  crystals  are 
easily  obtained. 

With  the  blood  of  the  rat,  all  that  is  needed  is  to  take  a  drop  of 
fresh  blood,  place  it  on  the  centre  of  a  glass  slide,  add  a  drop 
of  distilled  water,  and  when  the  edges  begin  to  dry,  cover 
with  a  cover  slip  and  examine  under  the  microscope  (Funke's 
method). 

2.  Hsematin. — Haemoglobin  is  composed  of  a  pigment  united 
with  a  proteid  body  which  has  erroneously,  according  to  Schae- 
fer,  been  called  globin.    The  pigment  may  be  separated  from  the 
proteid  in  the  following  manner: 

To  some  defibrinated  blood  in  a  test  tube  add  a  few  drops  of 
KOH  solution  and  heat  gently.  The  solution  assumes  a  greenish- 
red  color.  Now  carefully  neutralize  by  adding  dilute  HC1  until 
the  haematin  is  thrown  down  as  a  brownish  precipitate. 

The  same  result  is  attained  through  treatment  of  the  blood  with 
an  acid  and  then  neutralizing  with  an  alkali.  Haemoglobin  is 
therefore  decomposed  by  acids  and  alkalies  into  pigment  and 
albuminous  compounds.  All  the  iron  is  contained  in  the  hae- 
matin. 

3.  Haematin  Hydrochloric!  (haemin). — Place  a  very  small 
drop  of  blood  upon  a  glass  slide.  Mix  with  this  a  drop  of  glacial 
acetic  acid.  Heat  to  the  boiling  point  over  a  small  flame.  Allow 
the  fluid  to  evaporate  and  examine  the  residue  under  the  micro- 
scope. Tiny  reddish-brown  prismatic  crystals  will  be  seen.  These 

[73] 


\ 


4 


LABORATORY  MANUAL  OF  PHYSIOLOGY. 

are  also  known  as  Teichman's  crystals,  after  their  discoverer  (see 
Fig.  26). 

With  dried  blood  as  found  in  old  blood  stains,  a  small  crystal  of 
NaCl  is  added  to  the  acetic  acid  before  boiling. 

This  is  a  good  test  for  the  detection  of  blood  stains,  but  does  not 
identify  the  source  of  the  blood. 

4.  Haematoporphyrin. — This  is  derived  from  the  pigment 
portion  of  the  haemoglobin,  haematin,  by  a  splitting  off  of  the  iron 
radical. 

Mix  some  dried  blood  in  a  test  tube  with  concentrated  sulphuric 
acid.  Filter  the  resulting  solution  through  asbestos.  Divide  the 

filtrate  into  three  portions.  To 
the  first  add  an  excess  of  dis- 
tilled water.  To  the  second 
add  a  weak  solution  of  NaOH 
until  the  solution  is  slightly 
alkaline  in  reaction.  To  the 

NU     ^  ^i^^  third  add  an  excess  of  acidu- 

^   ^^  lated  alcohol. 

^^  The  water  in  the  first  solu- 

FIG.  26.— Haemin  Crystals.    (Frey.)  .  M1  .    .  ,       , 

tion  will  precipitate  the  haema- 

toporphyrin  as  a  brown  flocculent  mass.     The  iron  of  the  haematin 
unites  with  the  acid  to  form  a  ferrous  salt. 

In  the  second  tube  the  pigment  goes  into  solution.  In  the  third 
tube  a  solution  is  also  formed.  The  alkaline  solution  is  of  a  fine 
red  tint  changing  to  a  violet  in  the  presence  of  an  excess  of  the  re- 
agent. The  alcoholic  solution  has  a  purple  color,  changing  to  a 
bluish  violet  when  strongly  acidulated.  These  solutions,  accord- 
ing to  Schaefer,  exhibit  a  magnificent  red  fluorescence,  even  when 
exceedingly  dilute.  This  pigment  occurs,  in  small  amounts,  in  the 
normal  urine,  and  in  larger  quantities  in  certain  pathologic  condi- 
tions, such  as  chronic  sulphonal  poisoning.  It  is  also  closely  re- 
lated to  bilirubin,  a  bile  pigment,  and  haematoidin  (Virchow), 
which  is  found  in  old  blood  clots  within  the  body. 


[74] 


BLOOD. 

VIII.  SPECTRA  or  HEMOGLOBIN  AND  ITS  COMPOUNDS. 

A  convenient  instrument  to  use  is  the  micro-spectroscope  or 
spectroscopic  ocular  which  fits  into  the  tube  of  the  microscope  in 
place  of  the  ordinary  eyepiece. 

First  study  the  solar  spectrum.  With  a  narrow  slit  identify  the 
Fraunhofer  lines. 

1.  In  a  darkened  room  place  a  Bunsen  flame  in  line  with  the  re- 
flecting mirror  of  the  microscope  so  that  the  light  from  any  body 
made  luminous  in  the  flame,  will  pass  through  the  prisms  of  the 
spectroscope.    Dip  a  platinum  wire  in  a  solution  of  sodium  chlorid 
and  heat  in  the  flame.    What  is  the  nature  of  the  resulting  spec- 
trum?   Compare  this  with  the  spectrum  of  sunlight  or  the  light 
frcm  a  Welsbach  burner.    What  part  of  the  spectrum  is  luminous  ? 
To  what  absorption  band  or  Fraunhofer  line  does  this  correspond  ? 

2.  Repeat  the  observations  with  other  metallic  salts,  such  as 
strontium,  potassium,  barium,  and  copper. 

3.  Make  a  two-per-cent  solution  of  eosin  in  water.    Place  this  in  a 
vial  and  clamp  in  the  holder  at  the  side  of  the  eyepiece.     Tilt  the 
mirror  until  light  passes  through  the  solution  and  prism.    Arrange 
the  substage  mirror  for  reflecting  sunlight  through  the  comparison 
prism  so  that  the  two  spectra  may  be  viewed  side  by  side.     The 
eosin  absorbs  certain  parts  of  the  spectrum.     What  are  the  ab- 
sorption bands  of  eosin  ? 

4.  Oxy-hsemoglobin. — Fill  the  test  vial  with  defibrinated  blood 
diluted  with  ten  volumes  of  distilled  water.     Place  in  the  holder 
of  the  spectroscope  and  note  what  part  of  the  spectrum  is  visible 
and  what  part  has  been  absorbed.    Increase  the  dilution  until 
more  and  more  of  the  spectrum  becomes  visible.     Increase  the 
dilution  until  the  absorption  bands  can  no  longer  be  distinguished 
and  the  whole  of  the  spectrum  is  visible. 

What  are  the  absorption  bands  of  oxy-haemoglobin?  In  what 
part  of  the  spectrum  do  they  occur  ?  Make  a  drawing,  comparing 
the  absorption  spectrum  of  oxy-haemoglobin  with  the  solar  spec- 
trum. (See  text-book  for  pictures  of  various  spectra.) 

[75] 


LABORATORY  MANUAL  OF  PHYSIOLOGY. 

5.  Reduced    Haemoglobin. — (a)   Add  to  a  dilution  of  de- 
fibrinated  blood   in  which  the  two  absorption  bands  distinctly 
show,  a  few  drops  of  ammonium  sulphid.    Warm  gently.   The 
solution  changes  color,  becoming  purple  or  wine  color.    Examine 
the  spectrum  of  this  solution  and  compare  it  with  that  of  oxy-haemo- 
globin.     Open  the  vial  containing  the  reduced  haemoglobin  solu- 
tion, and  shake  it  vigorously  with  air.      Does  the  solution  change 
color?    Observe  the  spectrum.    Has  it  changed?    Is  this  change 
permanent  or  only  temporary  ? 

(b)  Dilute  the  solution  still  further.  Does  the  broad  absorp- 
tion band  resolve  into  several  or  does  it  simply  fade  as  the  blood 
becomes  more  dilute? 

6.  CO-haemoglobin.— (a)  Through  a   dilute  solution  of   de- 
fibrinated  blood,  showing  the  absorption  bands  of  oxy-haemoglo- 
bin,  pass  a  stream  of  CO  gas.    Note  the  characteristic  change  in 
color  from  the  bright  scarlet  of  oxy-haemoglobin  to  the  cherry  red 
of  CO-haemoglobin. 

(b)  Test  the  illuminating  gas  of  the  laboratory  for  carbon  mon- 
oxide in  this  way. 

(c)  Observe  the  spectrum  of  the  blood  so  treated.     Does  it  dif- 
fer markedly  from  that  of  oxy-haemoglobin  ?    Make  a  sketch  show- 
ing the  relations  of  the  absorption  bands. 

(d)  Add  a  reducing  agent,  as  was  done  in  obtaining  reduced 
haemoglobin.    Is  there  any  effect  upon  the  color  of  the  solution  or 
upon  its  spectrum  ?   What  conclusion  can  you  draw  concerning  the 
stability  of  this  haemoglobin  compound  ?    How  does  it  compare  in 
stability  with  oxy-haemoglobin?     Why  is  coal-gas  poisoning  so 
dangerous  ? 

7.  Methsemoglobin. — To  a   medium  dilute  solution  of  de- 
fibrinated   blood,    showing   the   two   absorption    bands   of   oxy- 
haemoglobin,    add    a    few    drops    of     potassium-permanganate 
solution.     Observe  the  spectrum.      If  the  oxy-haemoglobin  bands 
still  persist,  add  more  of  the  permanganate  and  warm  gently. 
Acidify  the  solution  and  look  for  the  spectrum  of  methaemo- 
globin. 

[76] 


BLOOD. 

To  the  solution,  add  some  reducing  agent.  Can  reduced  hae- 
moglobin be  obtained  from  methaemoglobin  ? 

8.  Hsematoporphyrin  (haematin  freed  from  iron). — Add  con- 
centrated  sulphuric  acid  to  some  defibrinated  blood  in  a  test 
tube.     Filter  through  asbestos.     Examine  the  spectrum  of  this 
solution. 

9.  Haematin. — Prepare    solutions    of    acid    haematin,     alkali 
haematin,  ethereal  solutions,  acid  alcohol  solutions.     Observe  the 
spectra,  comparing  them  with  each  other  and  with  the  spectra  of 
the  other  haemoglobin  derivatives,  studied  above. 

Drawings  should  be  made  of  all  the  absorption  spectra  that  you 
have  seen,  and  these  should  be  compared  with  the  table  of  spectra 
in  the  text-book  or  hanging  in  the  laboratory. 

IX.  GLOBULICIDAL  ACTION  OF  SERUM. 

1.  Mix,  in    a    small    test    tube,   equal   quantities  of  rabbit's 
blood  and  dog's  blood  serum.     Let  this  stand  for  twenty  to 
thirty  minutes  and  then  observe.     Is  there  any  change  in  the 
color   of    the    mixture?      Observe    with    reflected    and     trans- 
mitted   light.     Compare  with  blood  that  has  been  diluted  with 
water.     Place  a  drop  on  a  slide  and   examine  with    the  micro- 
scope.    Compare  this  with  a  fresh  sample  of  undiluted  rabbit's 
blood. 

2.  Heat  the  dog's  serum  to  60°  C.  for  ten  to  fifteen  minutes 
and  repeat  experiment  i. 

3.  Repeat  experiment  i,  using  rabbit's  serum  in  place  of  dog's 
serum,  and  dog's  blood  in  place  of  rabbit's  blood.    Do  the  same 
phenomena  occur  ? 

The  sera  of  certain  animals,  when  mixed  with  the  blood  of  cer- 
tain other  species,  cause  a  destruction  of  the  cellular  elements  with 
a  consequent  escape  of  the  haemoglobin  and  its  solution  in  the 
liquid  portion  of  the  mixture;  or,  in  other  words,  laking  occurs. 
This  property  is  true  for  other  cells  than  blood,  so  that, 
broadly,  it  is  known  as  cytolysis.  More  specifically,  in  the  case 
of  blood,  it  is  known  as  haemolysis,  and  the  serum  causing  such 

[77] 


LABORATORY  MANUAL  OF  PHYSIOLOGY. 

changes  is  said  to  be  lytic  for  those  particular  cells  upon  which  it 
acts. 

It  has  been  found  that  a  serum  which  is  not  normally  lytic  for 
the  blood  of  a  certain  animal,  may,  through  a  so-called  adaptive 
process,  be  made  to  acquire  such  properties.  This  may  be  de- 
monstrated in  the  following  series  of  experiments : 

4.  The  serum  of  the  guinea-pig  is  not  normally  lytic  for  the  cells 
of  rabbit's  blood.    Verify  this  statement  by  mixing  equal  parts  of 
rabbit's  blood  and  guinea-pig  serum  and  examining,  microscopi- 
cally, for  laking. 

5.  A  guinea-pig  is  adapted  for  rabbit's  serum  in  the  following 
manner:  5  c.c.  of  rabbit's  blood  is  injected  into  the  abdominal  cav- 
ity of  a  guinea-pig  every  third  day  until  five  injections  have  been 
given.     The  animal  should  be  weighed,  daily,  and  the  tempera- 
ture recorded.      If  the  reaction  following  the  injection  be  too 
severe,  the  quantity  must  be  reduced.    The  quantity  of  the  injec- 
tion may  then  be  increased  providing  that  there  is  no  decrease  in 
weight  or  marked  variation  in  temperature,  and  continued  until 
ten  injections  are  completed. 

The  pig  is  then  bled,  the  blood  allowed  to  clot,  and  the  serum 
collected. 

Bleed  a  rabbit.  Collect  the  blood  in  a  test  tube  containing  three 
times  the  volume  of  o.8-per-cent  NaCl  solution. 

(a)  Take  a  small  portion  of  the  diluted  blood  and  add  the  same 
amount  of  the  adapted  pig's  serum. 

(b)  To  a  second  portion  of  rabbit's '  blood  add  equal  parts  of 
guinea-pig's  serum  which  has  not  been  adapted. 

(c)  To  a  third  portion  of  blood,  add   some  of  the  adapted 
serum  which  has  been,  previously,  heated  to   60°  C.  for  thirty 
minutes. 

(d)  To  a  fourth  portion  of  blood  add  equal  parts  of  the  adapted 
serum  which  has  been  heated  and  serum  from  a  guinea-pig  which 
has  not  been  adapted. 

Compare  the  dilute  rabbit's  blood,  untreated,  with  tube  (a),  tubes 
(&),  (c),  and  (d).  Examine  slides  from  each  under  the  microscope. 

[78] 


BLOOD. 

Does  laking  occur  in  (a)  ?  Does  it  occur  in  (b)  ?  In  (c)  ?  In  (d)  ? 
Are  there  any  other  phenomena  observed  aside  from  laking? 
What  is  the  effect  of  heat  upon  the  adapted  serum  ?  How  can  the 
combined  action  of  heated  adapted  serum  and  unadapted  serum 
be  explained? 


[79 


CHAPTER  V. 
CIRCULATION  OF  THE  BLOOD. 

I.  STUDY  OF  THE  CIRCULATION  IN  THE  WEB  OF  THE 
FROG'S  FOOT. 

1.  INJECT  a  few  drops  of  a  one-per-cent  solution  of  curare  in 
the  dorsal  lymph  sac  of  a  frog.     After  fifteen  minutes  or  a  half 
hour  tie  the  frog,  face  down,  upon  the  frog-board.     Spread  the 
web,  but  not  too  tightly,  over  the  opening  in  the  board.    Place  the 
board  on  the  stage  of  the  microscope  so  that  the  opening  in  the 
board  coincides  with  the  opening  of  the  stage.    Study  the  circula- 
tion in  the  web  with  the  low  power  first.     If  the  circulation  in  the 
smaller  vessels  has  stopped,  the  web  is  drawn  too  tight  and  must  be 
somewhat  more  relaxed. 

Note  the  direction  of  the  flow  from  the  larger  vessels  into  the 
smaller  ones  and  from  the  smaller  toward  the  larger.  Can  you 
make  out  a  pulsation  in  any  of  the  vessels  ?  If  so,  in  which  ones  ? 
What  is  the  direction  of  the  flow  in  the  pulsating  vessels,  from 
large  vessel  into  branches  or  from  branches  into  large  vessel? 
What  is  the  speed  of  flow  in  the  large  vessels  as  compared  with  the 
smaller  ones  ?  Can  you  make  out  the  outlines  of  the  corpuscles  in 
any  of  the  vessels?  Can  you  distinguish  more  than  one  kind  of 
corpuscle  ? 

2.  Examine  now  with  a  higher  power,  so  that  the  corpuscles 
may  be  distinctly  seen.    Select  a  small  vessel  where  the  flow  is  not 
so  rapid  and  note  the  position  and  speed  of  movement  of  the  red 
and  white  corpuscles  in  the  Wood  stream. 

As  determined  by  the  movement  of  the  corpuscles,  in  what  part 
of  the  stream  is  the  speed  of  flow  greatest  ?  Explain. 

How  do  the  red  and  white  corpuscles  compare  in  numbers  ? 

[so] 


CIRCULATION  OF  THE  BLOOD. 

In  the  smaller  and  narrower  vessels  where  only  one  corpuscle  at 
a  time  can  get  through,  note  the  adaptability  of  the  red  cells, 
through  their  elasticity,  to  the  varying  calibre  of  the  vessel. 

3.  The  Migration  of  the  Leucocytes. — Dip  the  point    of  a 
pin  into  strong  acetic  acid.    Touch  the  web  with  the  acidulated  pin 
point.    Note  the  ensuing  effect  upon  the  circulation  through  the 
web  as  a  whole  and  particularly  upon  that  part  in  the  immediate 
vicinity  of  the  irritant. 

Examine  a  small  vessel  with  the  high  power.  How  does  the  rela- 
tive number  of  reds  and  whites  compare  with  the  relative  number 
in  the  non-irritated  web  ? 

Find  a  portion  of  the  web  free  from  pigment,  where  a  capillary 
may  be  seen  whose  walls  are  distinctly  visible.  Pick  out  some  leu- 
cocyte lying  against  the  wall  of  the  vessel  and  observe  it  closely. 
If  a  good  field  has  been  selected,  the  corpuscle  will  be  seen  to  make 
its  way,  gradually,  through  the  capillary  wall.  Make  a  series 
of  sketches  showing  the  progress  of  the  corpuscle  through  the  wall 
until  it  is  entirely  outside  of  the  vessel.  After  a  time  many  white 
cells  will  be  found  outside  of  the  vessels  in  the  surrounding  tissue. 
This  process  is  known  as  the  migration  of  the  leucocytes  and  oc- 
curs in  other  inflammatory  conditions  where  the  specific  irritant  is 
some  micro-organism  or  bacterial  toxin. 

Where  the~resulting  inflammation  is  more  severe,  the  vessel  walls 
may  so  change  as  to  allow  the  passage  of  the  red  cells  as  well  as  that 
of  the  whites.  The  white  cells,  however,  pass  through  the  vessel 
wall  by  means  of  their  amoeboid  movement,  while  the  reds,  in  their 
passage,  are  entirely  passive,  being  forced  through  by  the  pressure 
of  the  blood  in  the  vessel. 

4.  Repeat  the  observation  of  the  capillary  circulation,  using  the 
frog's  mesentery  instead  of  the  web  of  the  foot. 

The  mesentery  is  so  sensitive  that  simple  exposure  to  the  air  acts 
as  a  sufficient  irritant  to  cause  an  exhibition  of  all  the  phenomena 
of  inflammation. 

Make  a  drawing  of  the  capillary  circulation  as  seen  in  the  mes- 
entery and  in  the  web  of  the  frog's  foot. 
6  [Si]. 


LABORATORY  MANUAL  OF  PHYSIOLOGY. 


II.  DIRECT  OBSERVATION  OF  THE  ACTION  or  THE  FROG'S 
HEART. 

Pith  the  frog  used  in  the  previous  experiment  or,  if  necessary, 
take  a  fresh  frog.  Lay  the  frog  on  its  back  upon  the  cork  frog- 
board,  spreading  the  fore  legs  out  and  pinning  them  to  the  board. 
Do  the  same  to  the  hind  legs.  Make  a  median  incision  through  the 
skin  of  the  thorax,  crossing  this  transversely  with  an  incision  at  the 
level  of  the  fore  limbs.  Lay  back  the  flaps  of  skin  and,  with  small 


v.  D. 

FIG.  27. — Frog's  Heart.     V,  Ventral  view;  D,  dorsal  view.    B.A,  Bulbus  arteriosus  ; 
S.V,  sinus  venosus. 

but  strong  scissors,  continue  the  median  incision  through  the  tho- 
racic muscles  and  sternum.  Be  careful  to  keep  the  lower  blade  of 
the  scissors  snugly  against  the  inner  surface  of  the  sternum  in  order 
to  avoid  injury  to  the  pericardium  and  heart.  Hook  back  the  di- 
vided sternum  so  as  to  expose  the  heart  in  the  pericardium. 

Note  the  relations  of  the  pericardium  to  the  heart  and  great  ves- 
sels and  to  the  surrounding  viscera.  Keep  the  preparation  moist 
with  o.6-per-cent  NaCl  solution. 

Before  opening  the  pericardium,  note  the  rate  of  the  heart  beat, 
counting  the  number  of  beats  in  a  minute. 

Now  open  the  pericardium  so  as  to  obtain  a  better  view  of  the 
different  pulsating  portions  of  the  heart  and  make  the  following 
observations : 

[82] 


CIRCULATION  OF  THE  BLOOD. 

(a)  The  different  contracting  parts  (see  Fig.  27)   and  the  se- 
quence of  contraction. 

(b)  The  change  in  form  of  the  different  contracting  parts. 

(c)  The  change  in  color  of  the  different  parts  during  contrac- 
tion as  compared  with  relaxation. 

(d)  The  duration  of  the  systolic  period  as  compared  with  the 
diastolic  phase. 

(e)  The  change  of  position  of  the  heart,  as  a  whole,  with  each 
systole. 

(/)  Gently  grasp  the  ventricle  between  the  thumb  and  first 
finger  and  note  the  hardening  of  the  muscle  with  each  systole. 

(g)  Now  carefully  excise  the  heart,  including  all  its  pulsat- 
ing parts,  i.e.,  the  sinus  venosus  with  the  large  veins  which  empty 
into  it,  and  the  bulbus  arteriosus  with  pieces  of  the  arteries  into 
which  it  branches.  Place  the  excised  heart  in  a  shallow  dish  or  a 
watch  glass  containing  o.6-per-cent  NaCl  solution.  Does  the  heart 
continue  to  beat  ?  Is  the  normal  sequence  of  contraction  of  the 
different  parts  still  continued  ?  What  conclusion  can  you  draw 
from  this  observation  concerning  the  dependence  of  the  heart  beat 
upon  the  central  nervous  system  ? 

Count  the  number  of  heart  beats  per  minute  and  compare  with 
the  rate  of  pulsation  before  excision. 

(h)  Warm  the  heart  above  the  surrounding  room  temperature,        ^ 
by  holding  the  containing  vessel  in  the  hand.     How  is  the  beat 
affected  ? 

Float  the  watch  glass  on  cold  water  or  set  it  on  some  snow.  How 
is  the  rate  of  pulsation  affected  compared  with  the  normal  and 
with  that  of  the  warmed  heart  ? 

(i)  Using  the  same  heart  or  a  fresh  one  if  necessary,  cut  off  the 
sinus  from  its  connection  with  the  auricle.  Does  the  sinus  continue 
to  beat  ?  Do  the  auricles  and  the  ventricle  continue  to  beat  ?  If 
so,  is  there  any  difference  in  rate  of  pulsation  between  the  different 
parts? 

(y)  Sever  the  auricles  from  the  ventricle  by  an  incision  through 
the  auriculo-ventricular  groove.  Note  results  as  before. 

[83] 


LABORATORY  MANUAL  OF  PHYSIOLOGY. 

(£)  Separate  the  auricles  from  each  other.    Record  results. 

(/)  If  the  ventricle  has  stopped  beating,  see  if  it  will  still  re- 
spond to  mechanical  or  electrical  stimuli. 

Draw  conclusions  from  the  above  observations  concerning  the 
mechanism  of  the  heart-beat. 

EH.  GRAPHIC  RECORD  OF  THE  FROG'S  HEART-BEAT. 

Pith  a  frog.  Pin,  back  down,  on  the  frog  board.  Expose  the 
heart  as  in  the  previous  experiment.  The  heart  may  be  connected 
with  the  heart  lever  by  one  of  two  ways. 

1.  The  Suspension  Method. — Make  a  small  hook  out  of  a 
bent  pin.     Pass  this  through  the  tip  of  the  ventricle,  avoiding,  if 
possible,  piercing  the  cavity  of  the  ventricle.    Attach  this  hook,  by 
means  of  a  fine  thread,  to  the  short  arm  of  the  heart  lever,  placing 
sufficient  counterpoise  upon  the  long  arm  to  balance  the  weight  of 
the  thread  and  pin  and  to  raise  the  heart  slightly  from  its  bed. 
Apply  the  writing  point  of  the  lever  to  the  smoked  paper  of  a 
medium  fast  drum.    Also,  beneath  the  heart  record,  make  a  time 
tracing  in  seconds. 

The  auricular  beat  may  be  recorded  separately,  at  the  same 
time,  by  attaching  the  auricle,  in  the  same  way,  to  another  lever 
which  may  be  adjusted  to  write  under  or  over  the  ventricle  lever. 

2.  Direct    Transmission   Method. — An    upright,    made    of 
bamboo  or  some  other  light  material,  is  attached  to  the  long  arm 
of  the  lever  nearer  to  or  farther  from  the  axis,  depending  upon  the 
magnification  desired.    The  lower  end  of  the  upright  is  supplied 
with  a  cork  foot  which  may  be  made  to  rest  upon  the  auricle  or 
ventricle.    Every  movement  of  the  heart  chamber  upon  which  the 
foot  of  the  upright  rests  will  be  transmitted  to  the  long  arm  of  the 
lever  and  will  be  recorded  upon  the  revolving  drum. 

By  the  use  of  either  one  of  these  methods  record  the  contrac- 
tions of  the  ventricles  and  auricles,  noting  the  rate  of  pulsation  and 
the  duration  of  each  phase.  Also  note  the  form  of  the  curve  ob- 
tained during  systole.  Compare  systole  with  diastole. 

With  the  suspension  method  the  ventricular  record  will  prob- 

[84] 


CIRCULATION  OF  THE  BLOOD. 

ably  include  also  a  tracing  of  the  auricular  contraction  and,  if  the 
counterpoise  is  delicate  enough,  the  sinus  may  likewise  be  included. 

Note  the  relation  and  sequence  of  contraction  of  sinus,  auricles, 
and  ventricle. 

With  direct  transmission,  the  relations  of  auricular  pulsation  to 
ventricular  systole  and  diastole  may  be  observed  by  placing  the 
foot  of  the  writing  lever  upon  the  auriculo-ventricular  groove. 

The  relation  of  ventricle  to  bulbus  contraction  may  be  shown  in 
the  same  way,  by  adjusting  the  foot  of  the  lever  to  rest  partly  upon 
the  ventricle  and  partly  upon  the  bulbus. 

Make  careful  record  of  all  observations  and  interpretations  of  re- 
sults, marking  all  data  necessary  for  identification  upon  the  tracing. 

Compare  your  results  with  those  of  other  students  and  explain 
differences. 

What  part  of  the  tracing  is  due  to  errors  of  adjustment  of,  and 
inertia  of,  the  apparatus  ? 

IV.    INFLUENCE  OF  TEMPERATURE  UPON  THE   BEAT  OF  THE 
FROG'S  HEART. 

Use  the  same  heart  as  in  the  previous  experiment,  or,  if  this  is  not 
vigorous  enough,  make  a  fresh  preparation. 

Record  the  beat  of  the  ventricle,  taking  a  time  tracing  in  seconds 
or  half-seconds  in  order  to  determine  the  rate  of  pulsation. 

(a)  Determine  the  frequency  of  the  beat  at  the  room  tempera- 
ture, making  a  note  of  the  temperature  of  the  room. 

(6)  Bathe  the  heart  for  several  minutes  with  o.6-per-cent  XaCl 
solution  warmed  to  37°  C.  Allow  the  drum  to  revolve  again  at  the 
same  speed  as  before,  recording  the  second  tracing  under  the  first 
one  so  that  a  comparison  may  easily  be  made. 

(c)  Bathe  the  heart  again  with  salt  solution  at  the  room  tem- 
perature. Continue  the  bathing  with  salt  solution  cooled  in  an  ice 
bath  to  5°  C.  Make  another  record,  under  the  first  two,  together 
with  a  time  tracing. 

How  do  (a),  (6),  and  (r)  compare  in  the  number  of  beats  per 
minute? 


LABORATORY  MANUAL  OF  PHYSIOLOGY. 

How  do  the  contraction  curves  compare  with  each  other  ? 

How  does  the  time  ocupied  by  the  systolic  phase  compare  with 
the  time  occupied  by  the  diastolic  phase  in  each  tracing  ? 

When  the  frequency  of  the  heart-beat  is  increased,  is  the  time 
occupied  by  systole  and  that  of  diastole  decreased  in  the  same 
ratio  ? 

If  not,  which  is  shortened  the  more,  systole  or  diastole  ? 

V.  DISSECTION   OF   THE  EXTRINSIC  CARDIAC  NERVES  or  THE 

FROG. 

With  a  preserved  or  fresh  dead  frog,  expose  the  heart  as  before. 
Carefully  cut  away  the  sternum  and  muscles  of  the  thorax.  Locate 
the  glosso-pharyngeal  and  hypoglossal  nerves  (see  Fig.  28).  Also 


H 


FIG.  28. — Extrinsic  Cardiac  Nerves  of  Frog.  V,  Vago-sympathetic ;  Gp,  glosso- 
pharyngeal  nerve ;  Hg,  hypoglossal  nerve ;  Br,  brachial  plexus  ;  /.,  laryngeal  nerve ; 
//,  heart ;  Lv,  lung. 

note  the  petrohyoid  muscle.  Along  the  lower  border  of  this  muscle 
and  lying  between  the  loops  of  the  above-mentioned  nerves,  are 
two  fine  grayish  fibres.  These  are,  the  lower  one  the  vagus  trunk, 

[86]. 


CIRCULATION  OF  THE  BLOOD. 

containing  both  accelerator  and  inhibitor  fibres,  and  the  upper  one 
the  laryngeal  branch  of  the  vagus. 

Trace  the  cardiac  branch  of  the  vagus  trunk  to  its  distribution 
in  the  heart.  This  trunk  as  thus  exposed  in  the  thorax  is  really  a 
combined  nerve,  being  formed  by  the  junction  of  the  vagus  proper 
and  the  cardiac  branches  of  the  sympathetic. 

VI.  EFFECT  OF  STIMULATION  OF  THE  VAGO-SYMPATHETIC 
TRUNK  UPON  THE  HEART-BEAT. 

Pith  a  frog.  Carefully  expose  the  heart  and  isolate  the  vago- 
sympathetic  trunk.  Place  this  upon  fine  platinum  electrodes, 
avoiding,  so  far  as  possible,  contact  with  other  nerves  or  the  sur- 
rounding tissues. 

Connect  the  ventricle,  by  the  suspension  method,  with  a  light 
lever  made  to  write  upon  a  medium  fast  drum. 

(a)  Set  up    the  inductorium    for  weak  tetanizing  induction 
shocks  and  connect  the  secondary  with  the  vagus  electrodes.     Ar- 
range a  time-marker  writing  quarter-seconds  to  trace  beneath  the 
heart-beat  record. 

While  the  ventricular  tracing  is  being  taken,  stimulate  the  vagus 
with  weak  induction  shocks  for  ten  seconds.  Is  there  any  change 
in  the  rate  or  strength  of  the  heart-beat  ?  If  no  appreciable  effect  is 
produced,  increase  the  strength  of  the  current  slightly,  noting  the 
distance  of  the  secondary  from  the  primary,  and  repeat  the  stimu- 
lation of  the  nerve. 

(b)  Increase  the  strength  of  the  stimulus,  noting  the  effect  on 
the  heart-beat  with  each  increase,  until  strong  stimulation  is  em- 
ployed. 

What  is  the  effect  of  weak  stimulation  of  the  nerve  ?  Of  stronger 
stimulation  ?  Of  the  strongest  stimulation  which  you  applied  ? 

What  is  the  after-effect  upon  the  heart-beat,  following  the  cessa- 
tion of  the  stimulation  ? 

(c)  Apply  a  strong  stimulus  to  the  nerve  and  continue  this  for 
a  minute.    Does  inhibition  continue  during  the  entire  time  of  the 
application  of  the  stimulus  ? 


LABORATORY  MANUAL  OF  PHYSIOLOGY. 

Explain  the  difference  in  the  effect  of  strong  and  weak  stimu- 
lation. 

(d)  Effect  of  Stimulating  Vagus  Terminals. — Using  the  same 
heart,  stimulate  behind,  between  the  auricles  and  the  sinus  veno- 
sus.  Compare  these  results  with  those  of  (a),  (6),  and  (c). 

VII.  REFLEX  INHIBITION  OF  THE  FROG'S  HEART. 

Prepare  a  fresh  frog.  Do  not  pith,  but  anaesthetize,  lightly,  with 
ether.  Expose  one  sciatic  nerve  as  well  as  the  heart.  No  tracing 
need  be  made.  The  beat  may  be  studied  by  direct  observation. 

(a)  Count  the  number  of  beats  in  ten  seconds;  in  one  minute. 
With  a  scalpel  handle,  gently  tap  the  abdomen  in  the  stomach  re- 
gion for  five  seconds.  During  and  after  the  tapping,  observe  and 
count  the  number  of  heart-beats.  What  is  the  effect  of  the  tapping 
upon  the  beat  of  the  heart  ?  Is  it  accelerated  or  inhibited  ? 

(ft)  Introduce  into  the  stomach,  by  way  of  the  mouth  and 
oesophagus,  a  pair  of  shielded  electrodes.  In  this  way  stimulate 
the  stomach  with  a  medium  strong  current  and  note  results  in  rela- 
tion to  the  heart-beat. 

(c)  Cut  the  sciatic  nerve.    Is  there  any  effect  upon  the  heart- 
beat ?    Stimulate  its  central  end  with  a  medium  strong  current. 
What  is  the  effect  upon  the  heart-beat  ? 

(d)  Cut  both  vagus  nerves.    Can  reflex  inhibition  now  be  ob- 
tained ? 

VIII.  EFFECT  OF  DRUGS. 

^  1.  Atropine. — Pith  a  frog.  Expose  the  heart  and  vago-sym- 
pathetic  nerve.  Place  a  pair  of  small  electrodes  under  the  nerve 
and  connect  these  with  the  inductorium  arranged  for  medium 
strong  tetanizing  current.  Connect  the  ventricle  with  the  heart 
lever  for  recording  on  a  medium  fast  drum.  Take  a  normal  trac- 
ing, before  and  after  bathing  the  heart  with  physiological  salt  so- 
lution. Take  a  time  tracing  for  comparison.  Stimulate  the  nerve 
with  the  tetanizing  current  for  several  seconds.  There  should  be 
inhibition  of  the  heart-beat. 

Wait  until  the  after-effects  of  the  nerve  stimulation  have  disap- 


CIRCULATION  OF  THE  BLOOD. 

peared  and  then  bathe  the  heart  in  a  o.2-per-cent  solution  of  atro- 
pine  sulphate,  first  confining  the  application  of  the  drug,  so  far  as 
possible,  to  the  sinus  venosus.  Is  the  heart-beat  altered  in  any 
way? 

Now  repeat  the  stimulation  of  the  nerve.  What  is  the  effect  on 
the  heart-beat?  Is  there  acceleration  or  inhibition?  Compare 
this  with  the  tracing  obtained  before  the  application  of  the  drug. 

Stimulate  the  vagus  at  the  sinus.  Is  any  inhibitory  effect  pro- 
duced ?  What  effect,  then,  has  atropine  upon  the  extrinsic  nervous 
mechanism  of  the  heart  ? 

Continue  the  application  of  the  drug  until  its  effect  upon  the 
heart  muscle  becomes  manifest.  Wash  repeatedly  with  physiolog- 
ical salt  solution.  Does  the  heart  recover  from  the  effects  of  the 
atropine  ?  Repeat  the  stimulation  of  the  nerve.  Is  inhibition  again 
finally  brought  about  ? 

2.  Cocaine. — If  the  heart  used  in  experiment  i    has  recov- 
ered it  may  be  used  again  for  this  experiment.    Otherwise,  a  new 
preparation  will  have  to  be  made. 

The  heart  is  arranged  for  recording  as  before  and  the  vagus 
nerve,  for  stimulation.  A  control  tracing  is  made  of  the  heart-beat 
with  and  without  stimulation  of  the  nerve. 

While  the  tracing  is  being  taken,  allow  a  few  drops  of  a  0.2-per- 
cent solution  of  cocaine  in  o.6-per-cent  salt  solution  to  fall  upon 
the  heart.  Note  the  effect  upon  the  heart-beat.  Is  there  any  dis- 
turbance of  co-ordination  ? 

After  any  irregularity  in  the  beat  that  may  have  occurred  has 
disappeared,  stimulate  the  vago-sympathetic  nerve.  Is  any  inhib- 
itory effect  produced  ? 

Continue  the  application  of  the  cocaine  solution  until  the  ven- 
tricle ceases  to  beat.  Note  the  changes  in  the  heart's  action  as  the 
cocaine  effect  is  continued  and  increased. 

3.  Pilocarpine. — In  a  pithed   frog,  make   a   heart   prepara- 
tion for  recording,  and   expose  the  vago-sympathetic  nerve   for 
stimulating.    Take  first  a  tracing  of  the  beat  before  the  addition 
of  the  drug.    Make  another  tracing  during  stimulation  of  the  nerve 


LABORATORY  MANUAL  OF  PHYSIOLOGY. 

Now  bathe  the  heart  in  a  ten-per-cent  solution  of  pilocarpine  hy- 
drochlorate.  What  is  the  effect  upon  the  beat  ? 

Stimulate  the  nerve.  Is  there  any  inhibitory  effect  ?  If  so,  add 
more  of  the  pilocarpine  solution.  Stimulate  the  nerve  again.  If 
there  is  no  inhibition,  is  there  any  acceleration  ? 

After  the  heart  is  again  beating  slowly,  add  a  few  drops  of  weak 
atropine  solution.  What  is  the  result?  Add  more  pilocarpine. 
Does  the  atropine  effect  disappear  and  the  pilocarpine  effect  reap- 
pear ?  Repeat  this  alternate  treatment  with  pilocarpine  and  atro- 
pine, several  times.  Explain  the  action  of  pilocarpine  from  the  ob- 
servations thus  made.  Upon  what  does  the  antagonism  of  the 
two  alkaloids,  pilocarpine  and  atropine,  depend  ? 

4.  Muscarin. — Expose  the  heart  and  vago-sympathetic  nerve 
of  a  pithed  frog.    Take  a  tracing  of  the  normal  heart-beat.     Next, 
take  a  tracing  of  the  beat  while  stimulating  the  nerve. 

Bathe  the  heart  in  a  ten-per-cent  muscarin  solution.  Compare 
the  effect  upon  the  beat  with  the  pilocarpine  effect  and  with  the 
atropine  effect.  Stimulate  the  nerve.  Is  there  any  result  ?  Add  a 
few  drops  of  atropine  solution.  What  is  the  effect  ?  Explain. 

5.  Digitalin. — Prepare  a  frog  as  before.     Take  a  tracing  of 
the  normal  beat  and  of  the  beat  during  vagus  stimulation.    Apply 
to  the  heart  a  few  drops  of  a  saturated  solution  of  digitalin,  while 
a  tracing  is  being  taken.    Carefully  note  the  effect  upon  the  strength 
of  the  beat,  the  rate  of  beat,  and  the  change  in  systole  and 
diastole. 

Continue  the  application  of  the  alkaloid  until  the  heart  ceases  to 
beat.  Does  the  heart  stand  still  in  systole  or  diastole? 

6.  Nicotine. — Expose   heart   and   vagus   nerve    in   a    pithed 
frog.    Make  a  record  of  the  beat  before  and  during  stimulation  of 
the  nerve.    Note  the  character  of  the  normal  contraction.    Now 
bathe  the  heart  with  a  o.i-per-cent  solution  of  nicotine  in  physio- 
logical saline.    Continue  the  tracing  during  the  application  of  the 
drug.    What  is  the  effect  upon  the  frequence  and  strength  of  the 
heart-beat  ? 

While  the  heart  is  still  under  the  influence  of  the  drug,  stimulate 

[90] 


CIRCULATION  OF  THE  BLOOD. 

the  vago-sympathetic  nerve  with  a  strong  tetanizing  current.  Is 
there  any  inhibition  of  the  heart-beat  ? 

Stimulate  the  sinus  while  the  nicotine  effect  is  still  manifest.  Is 
there  any  inhibition  of  the  heart-beat  ?  How  can  you  explain  the 
difference  "in  result  between  stimulation  of  the  nerve  trunk  and 
stimulation  of  the  point  of  transfer  in  the  sinus  ?  Upon  what  part 
of  the  nervous  mechanism  does  the  nicotine  act  ? 

Now  bathe  the  heart  with  a  stronger  nicotine  solution  (one  per, 
cent).  Note  the  effect.  To  what  is  the  effect  due ? 

Paint  the  heart  with  the  pure  alkaloid.  In  which  phase  does  the 
heart's  action  cease  ? 

7.  Ether. — Expose  the  heart  of  a  pithed  frog.    Connect  with 
cardiograph  lever  and  take  a  tracing  of  the  normal  beat.     Shake 
up  some  ether  with  physiological  salt  solution  and  bathe  the  heart 
with  it,  while  a  tracing  is  being  taken.     Observe  the  effect  upon 
the  frequency  and  strength  of  the  beat.     Continue  the  application 
of  the  drug  and  the  observation  of  the  beat  of  the  heart. 

Wash  the  heart  with  undiluted  ether  until  the  beat  grows  feebler 
and  finally  nearly  ceases.  Wash  with  physiological  saline. 

Can  the  beats  be  restored?  Repeat  the  bathing  with  strong 
ether  until  the  beats  have  again  nearly  ceased  and  then  allow  a  few 
drops  of  a  i  to  10,000  adrenalin  solution  to  flow  on  the  heart. 
Result  ? 

8.  Chloroform. — A  frog  is  pithed,  as   before,  and  the  heart 
exposed.    A  tracing  of  the  normal  beat  is  taken  as  a  control.    A 
time  tracing,  in  seconds,  is  also  taken. 

Physiological  saline  is  shaken  to  saturation  with  chloroform. 
While  the  tracing  is  continued,  the  heart  is  bathed  with  this  mixt- 
ure. What  is  the  primary  effect  of  the  application  of  the  chloro- 
form? 

Continue  the  application  of  the  drug.  How  does  the  continua- 
tion of  the  chloroform  application  affect  the  heart-beat  ? 

Discontinue  the  application  of  the  drug  and  wash  the  heart  with 
physiological  salt  solution.  Does  the  heart  recover  from  the  effects 
of  the  chloroform  ? 

[91] 


LABORATORY  MANUAL  OF  PHYSIOLOGY. 

Bathe  again  with  chloroform,  this  time  undiluted.  Continue 
until  the  heart  stops  beating.  In  what  phase  does  the  stillstand 
occur?  How  does  the  chloroform  effect  compare  with  that  of 
ether?  How  does  the  heart-beat  change  in  frequency?  In 
strength?  In  length  of  diastole  and  systole?  To  which  is  the 
heart  more  susceptible,  chloroform  or  ether  ? 

9.  Suprarenal  Extract  (Adrenalin). — Take  a  tracing  of  a 
normally  beating  frog's  heart.  Make  up  a  i  to  10,000  solution 
of  adrenalin  chlorid  in  physiological  saline.  Bathe  the  heart 
with  this  solution  and  record  results.  What  is  the  effect  on  the 
strength  and  frequency  of  the  heart-beat?  Upon  systole  and 
diastole  ? 

Place  the  web  of  the  frog's  foot  under  the  microscope.  Locate 
certain  vessels  whose  outlines  are  quite  distinct.  Add  a  few  drops 
of  the  adrenalin  solution  to  the  web  and  note  the  effect  upon  the 
calibre  of  the  vessels. 

IX.  PERFUSION  OF  FROG'S  HEART. 

Pith  a  frog  and  expose  the  heart.  Excise  the  heart,  including  the 
sinus  venosus.  With  sharp-pointed  scissors  make  an  opening  in 
the  auricles.  Introduce,  through  this  opening,  Kronecker's  perfu- 
sion  cannula  into  the  ventricle.  Secure  the  cannula  by  means  of  a 
ligature  tied  above  the  base  of  the  ventricle.  Connect  one  limb  of 
the  cannula  with  the  perfusion  tube  and  the  other  with  the  small 
frog's-heart  manometer  (see  Fig.  29). 

Allow  the  heart  to  hang  in  the  normal  saline  bath.  Connect  this 
with  one  pole  of  a  dry  cell.  Connect  the  other  pole  with  the  bind- 
ing post  on  the  cannula.  Interpose  a  key  in  the  circuit.  The  heart 
may  stop  beating  for  several  minutes  after  the  cannula  is  tied  in. 
The  beats,  however,  will  generally  begin  spontaneously  after  a 
short  time.  If  not,  closing  the  key  of  the  constant  current,  and 
thus  stimulating  the  heart,  will  probably  be  sufficient  to  bring 
about  rhythmical  pulsations.  Simple  distention  of  the  ventricle 
with  the  perfusing  fluid  may  be  enough  of  a  stimulus. 

Fill  one  perfusion  tube  with  o.6-per-cent  NaCl  solution  and  the 

[92] 


CIRCULATION  OF  THE  BLOOD. 


-F- 


other  with  defibrinated  rabbit's  or  dog's  blood  diluted  with  equal 
volumes  of  o.6-per-cent  NaCl  solution. 

Perfuse  with  the  physiological  salt  solution.  From  time  to  time, 
close  the  outlet  tube  of  the  manometer  and  record  the  changes  in 
ventricular  pressure  on  a  revolving  drum  of  medium  speed.  Con- 
tinue the  perfusion  until 
the  rhythmical  beat 
ceases. 

Now  perfuse  with  the 
diluted  defibrinated 
blood.  Do  the  rhyth- 
mical pulsations  again 
begin  ? 

Repeat  with  a  fresh 
heart  preparation,  using 
the  diluted  blood  from 
the  start.  How  long 
are  rhythmical  contrac- 
tions continued  with  the 
blood  mixture  as  com- 
pared with  the  NaCl  so- 
lution alone  ?  Record 
the  pressure  changes  as 
before. 

Fill  one  perfusion  tube 
with  physiological  saline, 
saturated  with  ether. 
Perfuse  the  heart  with 
this  solution.  Record 
the  pressure  changes 
with  the  manometer. 

Compare  the  effect  of  ether  perfusion  with  that  obtained  through 
the  direct  application  of  the  drug. 

Change  the  perfusion  to  diluted  blood,  thoroughly  washing  out 
the  ether  solution.    Do  the  heart-beats  return  to  the  normal? 

[93l 


FIG.  29.  —  Frog's  Heart  Perfusion  Apparatus. 
(Kronecker.)  //,  Heart ;  C,  cannula ;  G,  glass 
containing  physiological  salt  solution;  E,  wires 
of  constant  current ;  F,  perfusion  flasks ;  Af, 
manometer. 


LABORATORY  MANUAL  OF  PHYSIOLOGY. 

If  so,  replace  the  ether  solution  with  one  of  chloroform  and  per- 
fuse with  the  latter.  Record  the  movements  of  the  manometer 
needle.  Compare  the  chloroform  effect  with  that  of  the  ether  and 
with  that  obtained  through  the  application  of  chloroform  to  the 
outside  of  the  heart. 

X.  THE  ACTION  or  CERTAIN  SALTS  ON  THE  HEART  MUSCLE. 

Expose  and  remove  the  heart  of  a  turtle.  Make  a  ventricular 
muscle  preparation  as  follows:  Make  two  cuts  through  the  ventri- 
cle, a  little  below  and  parallel  with  the  auriculo-ventricular  groove. 
The  cuts  thould  be  about  3  mm.  apart. 

The  ring  of  ventricle  thus  obtained  is  cut  through  at  opposite 
sides,  so  that  two  pieces  of  nearly  uniform  length  and  thickness  are 
obtained. 

Attach  one  end  of  each  strip,  by  means  of  a  fine  silk  thread,  to  the 
short  arm  of  a  light  counterpoised  muscle  lever.  The  other  end  is 
attached  to  one  limb  of  a  glass  rod,  bent  at  right  angles,  the  other 
limb  of  which  is  held  stationary.  The  lever  is  adjusted  to  the  sur- 
face of  a  slowly  revolving  drum.  Each  contraction,  therefore,  of 
the  muscle  strip  is  recorded.  By  the  time  the  preparation  is  com- 
plete, the  muscle  will,  probably,  have  ceased  beating. 

1.  Sodium. — (a)  Let  the  preparation  dip  in  a  beaker  containing 
a  0.7 -per- cent  solution  of  NaCl.    How  long  before  rhythmic  con- 
tractions of  the  muscle  strip  begin  ?    How  long  do  they  continue  ? 
What  are  the  rate  and  character  of  the  contractions  ?    Does  sodium 
act  as  a  stimulus  to  contraction  ?    Is  an  isotonic  sodium  solution 
sufficient  to  maintain  rhythmical  contractions? 

(6)  After  contractions  have  ceased  in  the  strip  immersed  in  the 
sodium  solution,  remove  the  strip,  blot  off  the  excess  of  the  solu- 
tion with  filter  paper,  and  immerse  in  another  beaker,  containing 
an  isotonic  calcium-chlorid  solution  (about  one  per  cent). 

(c)  Do  the  contractions  reappear  in  this  solution?  If  not,  im- 
merse again  in  the  sodium-chlorid  solution.  Do  contractions  now 
appear  ? 

2.  Calcium. — Immerse    another   strip  of   muscle  in  calcium- 

[94] 


CIRCULATION  OF  THE  BLOOD. 

chlorid  solution,  without  previous  immersion  in  the  sodium- chlorid 
solution.  Is  the  muscle  stimulated  to  pulsate  ?  Does  calcium  act 
as  a  stimulus  to  ventricular  contraction  ? 

3.  Combined  Action  of  Sodium  and  Calcium. — To  a  0.7- 
per-cent  solution  of  sodium  chlorid  in  a  beaker  add  one-tenth  of 
the  volume  of  calcium  chlorid.    Immerse  a  fresh  muscle  prepara- 
tion in  this  solution.    How  does  the  length  of  time  during  which 
contractions  are  maintained  compare  with  that  in  which  sodium 
chlorid  alone  was  used?    What  is  the  character  of  the  individual 
contractions  ? 

4.  Potassium. — Immerse  a  strip  of  ventricular  muscle  in  a 
o.Q-per-cent  potassium-chlorid  solution  which  is  nearly  isotonic 
with  0.7  per  cent  NaCl.     Are  rhythmical  contractions  brought 
about  ? 

5.  Combined  Action  of  Sodium,  Calcium,  and  Potassium. 
— Immerse  a  strip  of  ventricular  muscle  in  a  solution  of  sodium 
chlorid  0.7  per  cent,  calcium  chlorid  0.025   per  cent,   and   po- 
tassium  chlorid  0.025  per  cent  (slightly  modified  Ringer's  so- 
lution). 

Record  the  contractions  upon  a  very  slowly  moving  drum.  How 
long  will  rhythmical  contractions  continue?  How  do  they  com- 
pare with  those  obtained  from  the  muscle  treated  with  0.7  per 
cent  NaCl  alone,  and  with  those  from  the  muscle  immersed  in  a 
combination  of  Na  and  K  ? 

XI.  STANNIUS'  EXPERIMENT. 

Pith  a  frog.  Carefully  expose  the  heart.  Tie  the  fraenum,  the 
partition  of  pericardium  attached  to  the  dorsal  aspect  of  the  ven- 
tricle, and  use  the  ligature  as  a  guide.  Pass  a  thread  around  the 
junction  of  the  sinus  with  the  auricles  and  tie  snugly  (see  i,  Fig.  30). 

The  sinus  continues  to  beat.  The  auricles  and  ventricle  stop 
beating.  Now  tie  a  second  ligature  around  the  heart  at  the  auric- 
ulo-ventricular  groove.  The  ventricle  again  begins  to  beat.  The 
auricles  will  probably  remain  quiescent. 

Tie  a  third  ligature  about  the  middle  of  the  auricles,  at  line  2,  as 

[95] 


LABORATORY  MANUAL  OF  PHYSIOLOGY. 


indicated  in  the  figure.    The  auricles  will  again  begin  to  beat,  but 
at  a  different  rate  from  that  of  the  ventricle. 

Tie  a  fourth  ligature  about  the  base  of  the  ventricle  as  indicated 
by  line  4,  Fig.  30.    The  ventricle  will  again  cease  to  beat. 

XII.   MAXIMAL  RESPONSE  OF  HEART   MUSCLE    TO    MINIMAL 

STIMULUS. 

i .  Stop  the  rhythmical  contraction  of  a  frog's  heart  by  applying 
the  first  Stannius  ligature. 

Set  up  inductorium  for  single  induction  shocks.  Connect  tip  of 
ventricle  with  the  cardiograph  lever.  Arrange  the  drum  for  move- 
ment by  hand.  With  the 
secondary  coil  removed  as 
far  as  possible  from  the  pri- 
mary, apply  the  electrodes 
to  the  ventricle  and  break 
the  primary  circuit.  No  con- 
traction will  probably  occur. 
Move  the  secondary  nearer 
the  primary  and  repeat  the 
breaking  of  the  circuit,  at 
intervals  of  ten  seconds,  un- 
til a  stimulus  is  found  which 
will  cause  the  ventricle  to 
contract. 

Move  the  drum  slightly, 
increase  the  strength  of  stim- 
ulus, and  record  again. 
Repeat  with  stronger  and 
stronger  stimuli. 

The  contraction  in  re- 
sponse to  the  strongest  stimulus  is  no  greater  than  the  one  in 
response  to  the  weakest  stimulus  that  will  cause  a  contraction. 
The  heart  muscle,  therefore,  responds  to  a  minimal  stimulus  by 
a  maximal  contraction. 

[96] 


FIG.  30.— Schematic  Frog's  Heart,  to  Show 
Application  of  the  Stannius  Ligatures,  i, 
Between  sinus  (S)  and  auricles  G4);  2, 
middle  of  auricles;  3,  between  auricle  (A) 
and  ventricle  (V),  at  auriculo-ventricular 
groove;  4,  about  base  of  ventricle,  below 
groove. 


CIRCULATION  OF  THE  BLOOD. 

Compare  this  with  the  response  of  skeletal  muscle  to  stimuli  of 
various  strengths. 

XIII.  REFRACTORY  PERIOD. 

Connect  a  frog's  heart  with  the  cardiograph  lever.  Let  this  re- 
cord upon  a  medium  rapid  drum.  Arrange  a  signal  magnet  in  the 
primary  circuit,  with  the  point  of  its  writing  lever  placed  directly 
under  the  point  of  the  cardiograph  lever. 

Stimulate  the  ventricle  at  intervals  of  ten  or  fifteen  seconds, 
with  maximal  make  or  break  shocks  from  the  inductorium.  At- 
tempt to  stimulate  each  time  at  a  different  part  of  the  heart's  cycle. 

Some  of  the  stimuli  will  be  accompanied  by  an  extra-contrac- 
tion ;  others  will  have  no  effect  so  far  as  calling  forth  an  extra-ven- 
tricular contraction  is  concerned.  The  first  stimuli  were  applied 
during  the  irritable  stage  of  the  heart  muscle.  The  second  were 
applied  during  its  refractory  period.  Where  an  extra-contraction 
occurs,  it  is  followed  by  a  pause,  known  as  the  compensatory  paust, 
since  it  generally  restores  the  rhythm  which  prevailed  before  the 
extra-contraction  occurred. 

Systematically  stimulate  the  ventricle  at  different  periods  of  the 
cycle  and  note  your  results  on  the  drum,  thus  mapping  out  the 
limits  of  the  refractor}-  period. 

XIV.  DISSECTION  OF  MAMMALIAN  HEART. 

Open  the  thorax  of  a  dead  rabbit.  Note  the  position  of  the  heart 
in  the  pericardium  and  its  relation  to  the  surrounding  viscera. 
Note  the  reflections  of  the  pericardium  over  the  great  vessels  which 
enter  and  leave  the  heart.  Open  the  pericardium  and  note  the  po- 
sition of  the  heart  in  the  thorax.  What  part  of  the  heart  is  in  clos- 
est relation  to  the  chest  wall  ?  What  is  the  position  of  the  right  and 
left  ventricles  in  relation  to  the  anterior  thoracic  wall  ?  In  relation 
to  the  diaphragm  ?  Note  the  number  of  auricles  and  ventricles  in 
comparison  with  the  frog's  heart.  Also  note  the  more  distinct  dif- 
ferentiation of  the  pulmonary  system  in  the  mammal  as  compared 
with  the  frog. 

7  [97] 


LABORATORY  MANUAL  OF  PHYSIOLOGY. 

By  means  of  a  V-shaped  incision  through  the  walls  of  each  ven- 
tricle, with  the  apex  of  the  V  toward  the  apex  of  the  ventricle,  open 
the  right  and  left  ventricles.  Note  the  openings  between  the  auri- 
cles and  ventricles  and  between  the  ventricles  and  large  arteries. 
Note  the  shape  and  attachments  of  the  auriculo- ventricular  valves, 
the  chordae  tendineae  and  papillary  muscles.  Compare  these  with 
the  valves  guarding  the  openings  of  the  large  vessels,  the  semi- 
lunar  valves.  Determine  the  direction  of  opening  of  the  various 
valves. 

XV.  DISSECTION  AND  RELATIONS  OF  THE  EXTRINSIC  CARDIAC 

NERVES. 

This  should  first  be  done  upon  the  dead  animal.  Make  a  median 
incision  in  the  neck  of  a  rabbit,  through  the  skin  and  superficial 
fascia.  Continue  the  incision  down  to  the  trachea,  which  is  used  as 
a  guide.  Separate  the  muscles  from  either  side  of  the  trachea  and 
pull  them  to  one  side.  Beneath  the  sterno-mastoid  muscle  the  ca- 
rotid artery  and  the  large  nerve  trunks  running  along  with  it  will 
now  be  exposed.  Immediately  behind  the  artery  lies  the  vagus 
nerve  trunk.  This  nerve  contains,  among  a  number  of  other  fibres, 
inhibitory  fibres  for  the  heart.  A  somewhat  smaller  nerve  is  seen 
behind  and  to  the  inner  side  of  the  artery.  This  is  the  cervical 
sympathetic.  Next  to  this,  in  the  rabbit,  is  a  small  nerve,  the  de- 
pressor. Opposite  the  larynx,  the  superior  laryngeal  branch  joins 
the  main  vagus  trunk.  Here  also  the  depressor  nerve  joins  the  su- 
perior laryngeal  and  enters  the  main  trunk  with  it  or  splits  into  two 
branches,  one  joining  the  vagus  and  the  other  the  superior  laryn- 
geal (see  Fig.  31). 

XVI.  DIRECT  OBSERVATION    OF   THE  PULSATING  MAMMALIAN 

HEART.  , 

Inject  under  the  skin  of  a  rabbit  J  grain  of  morphine  sulphate. 
Lightly  anaesthetize  with  ether.  Expose  the  trachea  through  a  me- 
dian incision  in  the  neck.  The  rabbit  should  be  tied,  back  down, 
upon  the  rabbit-board,  the  fore  limbs  being  brought  down  to  the 

[98] 


CIRCULATION  OF  THE  BLOOD. 

sides  of  the  thorax  and  secured.  The  neck  should  be  well  stretched 
and  secured  in  the  head-holder.  A  simple  way  of  doing  this  is  to 
pass  a  short  thick  glass  rod  or  wire  nail  through  the  mouth,  behind 
the  teeth,  and  tie  about  the  jaws  and  to  the  board  behind  the  head. 


FIG.  31. — Extrinsic  Cardiac  Nerves  of  Rabbit.    C,  Carotid  artery;  V,  vagus  nerve; 
D,  depressor  nerve  ;  Sy,  sympathetic  nerve  ;  S.L,  superior  laryngeal  nerve. 

Open  the  trachea  by  a  transverse  incision.  Introduce  and  tie  in 
the  tracheal  cannula.  Connect  this  with  some  form  of  artificial 
respiratory  apparatus  and  start  artificial  respiration  at  about  the 
same  rate  that  the  rabbit  was  breathing  before. 

Make  a  median  incision  through  the  skin  of  the  thorax  down  to 
the  sternum  and  as  far  down  a&.its  lower  end.  With  heavy  scissors 


LABORATORY  MANUAL  OF  PHYSIOLOGY. 

or  bone  scissors  specially  made  for  the  purpose,  cut  through  the 
sternum,  keeping  the  lower  blade  of  the  scissors  closely  applied  to 
the  under  side  of  the  sternum.  With  care,  the  internal  mammary 
artery  will  probably  be  avoided.  If  it  is  cut,  it  must  be  secured 
with  artery  forceps  and  tied. 

The  artificial  respiration  should  be  stopped  during  the  operation 
of  opening  the  thorax,  in  order  to  avoid  injuring  the  lungs.  Start 
the  artificial  respiration  again.  Pull  the  divided  thorax  apart  and 
expose  the  pulsating  heart  in  the  pericardium.  Note  its  relations 
to  the  lungs  during  inspiration  and  expiration.  Stop  the  respira- 
tion for  a  moment  and  open  the  pericardium.  Note  the  character 
and  sequence  of  contraction  of  the  various  chambers.  Note  the 
change  of  position  of  the  heart  with  each  beat.  Compare  ventric- 
ular systole  with  diastole.  Note  the  coronary  arteries  and  veins  on 
the  surface  of  the  heart.  How  does  the  method  of  nutrition  of  the 
mammalian  heart  compare  with  that  of  the  frog  ?  Feel  the  heart 
between  the  thumb  and  finger.  Note  the  hardening  of  the  ven- 
tricle with  each  systole  and  its  softening  with  each  diastole. 

With  artificial  respiration  stopped,  note  the  change  in  the  rate 
of  the  heart-beat  as  asphyxia  continues,  also  the  distention  of  the 
right  ventricle.  Also  note  the  change  in  color  of  the  blood.  Begin 
respirations  again  and  note  the  recovery  of  the  heart  and  the  change 
in  the  color  of  the  blood. 

XVII.  STIMULATION  OF  EXTRINSIC  CARDIAC  NERVES. 

i.  In  the  same  rabbit  used  in  the  previous  experiment,  expose 
and  isolate  the  vagus  and  depressor  nerves.  Tie  both  nerves  be- 
tween ligatures,  Cut  between  the  ligatures.  Stimulate  the  distal 
end  of  the  vagus  with  a  strong  tetanizing  current  and  observe  the 
heart's  action.  Stimulate  the  central  end  of  the  divided  depressor 
and  observe  the  effect  upon  the  heart-beat.  Stimulate  the  distal 
end  of  the  depressor.  Is  there  any  change  in  the  beat  of  the  heart  ? 

Trace  the  cervical  sympathetic  down  into  the  thorax  to  the  stel- 
late ganglion.  Place  this  upon  the  electrodes  and  stimulate 
with  a  medium  strong  tetanizing  current.  What  is  the  effect 

[i.oo] 


CIRCULATION  OF  THE  BLOOD. 

upon  the  heart-beat  ?  Compare  this  with  the  strong  vagus  stim- 
ulation. 

Stimulate  the  ventricle  directly  with  a  tetanizing  current  and 
note  the  change  in  the  character  of  the  contractions.  Compare  the 
feel  of  the  heart  in  this  condition  with  that  of  the  normally  beating 
heart.  After  the  cessation  of  the  exciting  cause  of  the  fibrillary 
contractions,  the  rabbit's  heart  will  again  pulsate  normally.  The 
heart  of  the  dog  will  continue  to  fibrillate  until  death,  unless  it  is 
removed  and  perfused  with  defibrinated  blood  or  saline  solution. 

Excise  the  heart.  Does  it  still  continue  to  beat  ?  Sever  the  ven- 
tricles from  the  auricles  by  a  cut  below  the  auriculo-ventricular 
groove.  Do  the  severed  parts  continue  to  pulsate  ?  If  the  ventri- 
cles have  stopped,  are  they  still  irritable  to  mechanical  stimuli  ? 
Sever  the  auricles  from  each  other.  Do  they  still  continue  to  beat  ? 

XVIII.  ACTION  OF  THE  HEART  VALVES. 

The  following  simple  scheme  may  be  used  to  demonstrate  the 
action  of  the  semilunar  valves  (see  Fig.  32).  A  dog's  heart  or  a 


J-A 


FIG.  32.  —Apparatus  to  Show  Action  of  Semilunar  Valves.    (Description  in  text.) 

fresh  pig's  heart  nlay  be  used.    A  tube,  T,  with  a  smoothly  cut  end 
over  which  is  cemented  a  flat  piece  of  glass,  is  connected  through 

[I0t] 


LABORATORY  MANUAL  OF  PHYSIOLOGY. 

a  sidepiece  to  a  pressure  bottle,  A .  The  open  end  of  the  tube  is  in- 
serted into  the  aorta  or  pulmonary  artery,  above  the  semilunar 
valves,  and  tied  in.  The  ventricle  behind  the  valves  is  cut  away, 
enough  being  left  for  the  insertion  and  securing  with  a  snugly  tied 
ligature  of  another  glass  tube  which  is  connected  through  rubber 
tubing  with  another  pressure  bottle,  P,  used  for  exerting  pressure 
on  the  ventricular  side  of  the  valves.  Pressure  bottle  B  is  used 
for  the  filling  of  P,  and  can  be  clamped  off  when  this  has  been  ac- 
complished. 

Bottle  P  has  had  the  bottom  removed,  and  in  place  of  it  a  heavy 
rubber  dam  has  been  substituted.  By  raising  or  lowering  bottle 
A ,  the  pressure  on  the  arterial  side  of  the  valves  may  be  increased 
or  decreased.  The  movement  of  the  valves  may  be  observed 
through  the  glass  plate  of  tube  T. 

Make  sudden  pressure  with  the  hand  on  the  rubber  membrane 
of  bottle  P.  Note  the  opening  of  the  valve  flaps.  In  which  direc- 
tion do  they  open  ?  Note  their  closure  when  the  pressure  on  the 
arterial  side  is  greater  than  that  on  the  ventricular  side.  Increase 
the  arterial  pressure  by  elevating  the  bottle  A .  Note  the  increased 
pressure  needed  at  P  to  open  the  valves.  What  prevents  the  valves 
from  being  forced  back  into  the  ventricle  ? 

The  same  scheme  may  be  utilized  for  showing  the  action  of  the 
auriculo-ventricular  valves  by  tying  tube  T  into  the  auricle,  and 
the  tube  connected  with  bottle  P  into  the  ventricle,  with  the  aorta 
clamped  off. 

XIX.  MECHANICS  OF  THE  CIRCULATION  AS  STUDIED  WITH  AN 
ARTIFICIAL  SCHEMA. 

Set  up  an  artificial  scheme  of  the  circulation  as  shown  in  Fig. 
33.  H  represents  a  Davidson's  syringe  having  an  inlet  and  an 
outlet  valve  which  correspond  to  the  valves  between  the  auricles 
and  ventricles  and  between  the  ventricles  and  arteries,  respec- 
tively. The  fluid  is,  therefore,  allowed  to  flow  in  one  direction 
only.  A  represents  arteries,  C  capillaries,  and  V  veins.  Ma  is  a 
manometer  connected  with  an  artery  to  show  changes  in  arterial 

[102] 


CIRCULATION  OF  THE  BLOOD. 

pressure.  Mv  is  a  manometer  connected  with  a  vein  to  show 
changes  in  venous  pressure.  The  glass,  containing  water,  repre- 
sents the  auricle ;  the  bulb  of  the  syringe,  the  ventricle.  The  in- 


FIG.  33.— Artificial  Schema  of  the  Circulation.    (Description  in  text.) 

crease  in  resistance  in  the  circulation  that  occurs  in  the  capillary 
area  is  imitated  by  screw  clamps  on  the  tubing.  The  resistance 
may  be  increased  by  screwing  the  clamps  more  tightly,  and  de- 


LABORATORY  MANUAL  OF  PHYSIOLOGY. 

creased  by  loosening  the  clamps.  Tightening  the  screws  would 
imitate  arterial  constriction.  Loosening  them  would  correspond 
to  a  vaso-dilatation. 

Before  using  the  artificial  schema,  make  out  the  following  points. 
Connect  the  syringe  with  a  long  piece  of  glass  tubing.  When  the 
syringe  is  filled,  squeezing  it  with  the  hand  will  force  fluid  out 
through  the  glass  tube.  Release  of  the  bulb  will  cause  it  to  fill 
from  the  vessel  in  which  the  inlet  tube  is  dipped.  Press  the  bulb  of 
the  filled  syringe  with  the  hand.  Note  that  water  is  forced  through 
the  glass  tube  and  out  of  its  free  end  in  a  jet  which  ceases  as  soon 
as  the  pumping  force  behind  stops.  Squeeze  the  bulb  a  number 
of  times  in  succession.  Note  that  there  is  no  flow  from  the  tube 
between  pumps.  In  vessels  with  inelastic  walls  all  the  force  of  the 
pump  is  exerted  in  moving  the  column  of  fluid  forward  and  in 
overcoming  friction.  The  friction  is  inversely  proportional  to  the 
size  of  the  tube.  The  smaller  the  tube,  the  greater  the  friction. 
The  longer  the  tube,  the  greater  the  friction. 

For  the  glass  tube,  substitute  a  long  rubber  tube  of  small  calibre. 
Press  the  syringe  bulb  a  number  of  times  in  rapid  succession.  The 
water  will  still  spurt  from  the  open  end  of  the  tubing  with  each 
stroke  of  the  pump,  but  there  will  probably  be  some  flow  between 
strokes.  This  may  be  increased  and  the  flow  during  the  stroke  of 
the  pump  decreased  by  partly  clamping  the  tube  near  the  free 
end.  In  other  words,  the  peripheral  resistance  has  been  increased. 

If  the  peripheral  resistance  is  sufficiently  increased  the  flow  be- 
comes continuous.  With  elastic  tubes  and  a  high  resistance  to 
overcome,  part  of  the  force  of  the  pump  is  expended  in  distending 
the  walls  of  the  elastic  vessel.  When  the  distending  force  has 
ceased,  the  elastic  wralls  rebound  and  force  the  stored-up  fluid  on. 
With  each  stroke  of  the  pump,  then,  in  a  system  of  elastic  vessels 
some  of  the  energy  becomes  latent  in  the  distended  walls  of  the 
vessels,  to  be  transformed  into  kinetic  energy  in  the  interval  be- 
tween pumps. 

With  the  clamps  of  the  circulation  schema  open,  press  the  sy- 
ringe bulb  a  number  of  times  in  slow  succession.  Note  the  charac- 

[104] 


CIRCULATION  OF  THE  BLOOD. 

ter  of  the  flow  from  the  venous  end  of  the  schema.  Is  it  continuous, 
remittent,  or  intermittent?  Note  also  the  excursions  of  the  col- 
umn of  mercury  in  each  manometer.  Is  there  any  difference  in 
pressure  as  indicated  by  the  two  manometers  ?  Explain. 

Increase  the  peripheral  resistance  by  tightening  one  of  the  cap- 
illary damps.  Repeat  the  intermittent  pressure  on  the  syringe 
bulb.  Has  the  character  of  the  flow  changed  ?  Compare  the  two 
manometers.  Is  there  any  change  in  arterial  pressure  as  com- 
pared with  venous  pressure?  Is  the  fall  in  arterial  UMamic  be- 
tween the  strokes  of  the  pump  large  or  small?  Does  the  venous 
pressure  still  rise  and  fall  with  the  heart-beat  ?  FTplam. 

Increase  the  peripheral  resistance  still  more  by  tightening  more 
clamps.  Note  the  change  in  arterial  pressure  as  compared  with 
venous  pressure,  during  the  series  of  pump  strokes.  Note  the 
change  in  the  character  of  the  outflow  of  fluid  from  the  veins  and 
the  speed  of  flow  as  compared  with  the  speed  with  lower  resistance. 
Does  the  flow  continue  after  the  pump  has  ceased  to  act?  Does 
the  arterial  pressure  fall  more  slowly  or  more  rapidly  than  before? 

Is  the  excursion  of  the  column  of  mercury  in  the  arterial  manom- 
eter greater  or  less  with  each  pump  stroke  dm  before? 

Decrease  the  rate  of  pump  strokes.  What  is  die  effect  on  blood 
flow  and  on  arterial  pressure? 

Dilate  the  arterioles  by  loosening  a  damp.  Using  the  same  rate 
of  pump  strokes  as  before  and  as  nearly  as  possible  the  same 
strength  of  stroke,  what  is  the  effect  on  arterial  pressure  and  on 
venous  flow? 

Record  of  Pulse  in  Artificial  Schema. — Close  the  damps  on  the 
tubes  representing  the  capillary  drculation  until,  with  freqeunt 
regular  compressions  of  the  pump,  a  constant  outflow  from  the 
reins  is  obtained  and  die  asdOatiaas  of  die  arterial  mannmetfr 
are  slight.  Determine  the  mean  arterial  pressure  by  multiplying 
the  height  of  the  column  of  mercury  in  the  distal  limb  of  the  manom- 
eter above  the  meniscus  in  the  proximal  limb  by  2. 

Clamp  off  the  immaaHH  from  die  artery  tube.  On  the  artery 
near  the  bulb  adjust  a  receiving  tambour.  Connect  this  with  a  re- 


LABORATORY  MANUAL  OF  PHYSIOLOGY. 

cording  tambour,  whose  writing  lever  is  applied  to  the  smoked  sur- 
face of  a  medium-slow  drum.  The  increase  in  pressure  at  each 
stroke  of  the  pump  is  partly  employed  in  distending  the  vessel. 
This  causes  a  bulging  of  the  vessel  and  a  hardening  which  can  be 
felt  by  the  finger  and  which  in  this  case  is  transmitted  through  the 
receiving  tambour  to  the  recording  tambour  and  is  written  as  a 
curve  on  the  revolving  drum.  This  is  the  pressure  pulse  and  is 
indicative  of  the  rise  in  pressure  in  the  artery  with  each  systole  of 
the  ventricle.  The  lever  of  the  recording  tambour  should  be  deli- 
cately adjusted  to  the  surface  of  the  drum  so  as  to  reduce  friction 
to  a  minimum. 

What  is  the  form  of  the  pressure  pulse  in  this  instance?  How 
does  the  systolic  rise  compare  with  the  diastolic  fall?  Are  there 
any  secondary  waves  ?  If  so,  what  is  their  significance  ? 

Increase  the  rate  of  the  pump  stroke.  What  change  occurs  in 
the  pulse  wave?  Decrease  the  rate  of  the  pump  stroke.  What 
change  occurs  in  the  form  of  the  pulse  wave  ? 

Increase  the  peripheral  resistance  by  tightening  the  clamps  on 
the  capillaries.  What  is  the  effect  on  the  pulse?  Decrease  the 
peripheral  resistance.  What  is  the  effect  on  the  pulse  ? 

With  the  capillary  clamps  so  applied  that  the  venous  outflow  is 
continuous,  place  the  finger  upon  the  venous  tube  while  the  heart 
bulb  is  rhythmically  pressed.  Is  any  pulse  felt  in  the  veins  ?  Ap- 
ply the  receiving  tambour  to  the  vein.  Is  any  pulse  recorded  by 
the  recording  tambour  ? 

Release  the  compression  in  the  capillary  region  until  the  venous 
outflow  becomes  remittent.  Is  a  pulse  tracing  now  obtainable  from 
the  veins  ?  Explain. 

^kx.  PULSE  RECORD  IN  MAN. 

By  the  Tambour  Method. — A  simple  method  for  recording  the 
pulse  is  by  some  such  scheme  as  depicted  in  Fig.  34.  This  con- 
sists of  a  thistle  tube,  T,  to  act  as  a  receiving  tambour,  and  a  re- 
cording tambour,  R,  connected  with  the  receiving  tambour  by 
strong-pressure  tubing  for  transmitting  the  impulse  from  one  tarn- 

[106] 


CIRCULATION  OF  THE  BLOOD. 

bour  tp  the  other.  R  is  covered  with  a  thin  rubber  membrane, 
drawn  not  too  tightly,  against  which  rests  a  long  light  lever.  If 
the  carotid  pulse  is  taken,  it  is  not  necessary  to  cover  the  thistle 
tube  with  rubber.  The  integu- 
ment over  the  artery,  against 
which  the  tube  is  tightly  pressed, 
acts  as  such. 

If  a  record  of  the  radial  pulse 
is  to  be  taken,  the  thistle  tube 
should  be  covered  in  the  same 
way  as  the  recording  tambour,  T 

and  a  Cork  button  cemented  to    FIG.  ^.-Simple  Sphygmograph  and  Car- 

diograpn.  (Description  in  text.) 

the  middle  of  the  rubber  mem- 
brane. The  button  is  applied  to  the  integument  over  the  artery, 
the  movements  of  the  artery  are  transmitted  to  the  button  of  the 
receiving  tambour  and  its  rubber  membrane,  and  this  in  turn  is 
transmitted  to  the  membrane  and  lever  of  the  recording  tambour 
which  writes  the  record  upon  the  smoked  paper  of  the  revolving 
drum. 

Take  a  tracing  of  the  carotid  pulse,  adjusting  the  receiving  tam- 
bour until  a  definite  curve  is  obtained.  Make  out  the  general  rise 
and  fall  of  the  pulse  pressure  with  each  systole  and  diastole  and 
the  secondary  waves  of  the  tracing.  Explain. 

Repeat,  taking  a  record  from  the  radial  artery.  Compare  the 
sphygmographic  record  with  palpation  of  the  artery  by  the 
finger. 

Take  sphygmographic  records  with  the  spring  sphygmographs 
of  Marey  or  Dudgeon  or  some  other  similar  instrument.  Compare 
with  the  record  taken  by  the  tambour  scheme. 

XXI.  VOLUME  PULSE. 

The  pulse  record  as  taken  by  the  sphygmograph  measures  with 
a  fair  degree  of  approximation  the  changes  in  pressure  in  an  artery 
brought  about  by  ventricular  systole  and  diastole,  but  measures 
very  inexactly  the  changes  in  volume. 


LABORATORY  MANUAL  OF  PHYSIOLOGY. 

For  this  determination  a  larger  vascular  area  is  required.  An 
instrument  used  for  determining  volume  changes  is  called  a  ple- 
thysmo graph.  One  of  the  simplest  devices  is  that  of  Porter. 

This  consists  of  a  glass  tube,  with  a  rubber  collar  made  to  fit 
snugly  around  the  middle  finger.  The  tube  is  connected  with  a 
recording  tambour,  and  the  volume  changes  of  the  finger  are  re- 
corded upon  a  slowly  revolving  drum. 

These  changes  are  rhythmical  in  character  and  correspond  to 
the  rhythm  of  the  heart-beat.  In  addition,  larger  waves  may  be 
written  as  a  result  of  general  or  local  vasomotor  changes. 

A  larger  record  is  obtained  through  the  use  of  Mosso's  water 
plethysmograph,  by  which  the  volume  changes  of  the  forearm  are 
recorded. 

This  consists  of  a  large  glass  cylinder  provided  with  four  open- 
ings— one  for  the  insertion  of  the  forearm,  one  for  connection  with 
the  recording  apparatus,  one  for  filling  the  system  with  water,  and 
one  for  the  insertion  of  a  thermometer. 

A  simple  recording  device  is  the  water  pen  of  Kronecker.  This 
is  a  small  box  connected  by  rubber  tubing  with  the  plethysmo- 
graph cylinder  upon  the  surface  of  the  water  in  which  a  cork  sheet, 
supporting  a  writing  lever,  is  floated. 

The  hand  and  forearm  are  anointed  with  vaseline,  a  rubber  col- 
lar is  fitted  snugly  just  below  the  elbow,  the  hand  and  forearm  are 
inserted  in  the  cylinder,  and  the  rubber  collar  fitted  about  the 
flange  of  the  opening  through  which  the  arm  is  inserted.  The  cyl- 
inder is  connected  with  the  recording  apparatus,  the  cylinder  and 
recording  apparatus  are  filled  with  water,  the  thermometer  is  in- 
serted in  the  opening  provided  for  it,  the  filling  bottle  is  clamped 
off,  and  the  recording  pen  is  applied  to  the  surface  of  a  slowly  re- 
volving drum. 

Set  up  the  apparatus  as  described  above  and  take  a  normal- vol- 
ume pulse  tracing. 

While  the  tracing  is  being  recorded,  elevate  the  free  hand  and 
arm  above  the  head.  Is  there  any  change  in  volume  of  the  arm  in 
the  cylinder? 

[108] 


CIRCULATION  OF  THE  BLOOD. 

Make  a  series  of  rapid  shallow  respirations.  Is  there  any  change 
in  the  tracing  ? 

Make  a  series  of  deep  slow  respirations.  What  change  occurs 
in  the  tracing  ? 

Hold  the  breath  for  fifteen  seconds.  Note  any  change  in  the 
volume  tracing. 

While  the  tracing  is  being  taken,  work  out  some  mathematical 
problem.  What  is  the  effect  on  the  volume  of  the  arm  ? 

Immerse  th£  free  hand  in  ice  water.  This  will  cause  a  primary 
vaso-constriction  in  the  vessels  of  the  hand.  Is  there  a  corre- 
sponding vaso-constriction  in  the  other  hand,  as  indicated  by  a  fall 
in  volume  ? 

Allow  the  subject  of  the  experiment  to  take  a  few  whiffs  of  amyl 
nitrite.  This  is  a  general  vaso-dilatant.  What  is  the  effect  on  the 
plethysmographic  tracing  ? 


APEX  BEAT,  CARDIOGRAM,  AND  HEART  SOUNDS. 

1.  Let  a  student  strip  to  the  waist.    Locate  the  apex  impulse. 
What  is  its  relation  to  the  sternum?  to  the  nipple?  to  the  ribs 
and  intercostal  spaces  ?    How  does  its  position  vary  with  the  posi- 
tion of  the  subject  ? 

2.  Map  out  the  cardiac  area  on  the  chest  wall  —  (a)  with  light 
percussion;    (b)  with  heavy  percussion.     Mark  the  area  on  the 
chest  with  a  colored  pencil. 

3.  Prepare  a  cardiograph  as  follows:  Take  a  large  thistle  tube, 
about  five  centimetres  in  diameter,  stretch  a  rubber  membrane 
across  the  top  and  cement  a  cork  button  to  the  middle  of  the 
membrane.     Connect  this,  through  a  piece  of  pressure  rubber 
tubing,  with  a  recording  tambour  whose  diameter  should  be  some- 
what less  than  that  of   the  thistle  tube.     In  order   to   obtain 
sufficient   magnification,   the    recording   lever   should    be   about 
twenty-five  centimetres  long.     A  good  writing  point  may  be  cut 
out  of  a  piece  of  the  glazed  paper  used  for  tracings.  . 

Adjust  the  writing  point  of  the  recording  lever  to  the  smoked 
paper  of  a  medium-slow  drum.    Press  the  button  of  the  receiving 

[.09] 


LABORATORY  MANUAL  OF  PHYSIOLOGY. 

tambour  to  the  apex  impulse.  A  curve  will  be  written  on  the 
drum.  Make  a  number  of  tracings  from  the  same  individual, 
varying  the  pressure  of  the  cardiograph  against  the  chest  wall 
and  varying  the  position  of  the  subject  of  the  experiment. 

Compare  the  different  tracings  taken  from  the  same  individual 
with  each  other.  Does  the  curve  recorded  consist  of  a  single  wave 
or  of  a  wave  with  crests  and  depressions?  Note  the  notches  and 
apices  of  the  waves  which  occur  in  all  the  tracings  and  the  points 
in  the  curve  in  which  they  occur. 

Take  a  series  of  tracings  from  another  individual  and  compare 
the  essential  features  of  these  with  those  of  the  other  series. 

With  a  stethoscope  listen  over  the  cardiac  area  for  the  heart 
sounds.  Palpate  the  pulse  at  the  wrist  or  over  the  carotid  at  the 
same  time.  How  many  different  sounds  do  you  hear  ?  What  are 
their  characteristics  in  pitch,  duration,  and  synchronism  with  the 
pulse  wave  ? 

At  what  part  of  the  cardiac  area  is  the  first  heart  sound  best 
heard  ?  Where  is  the  second  sound  most  distinctly  heard  ?  What 
factors  enter  into  the  production  of  the  heart  sounds  ?  Where  can 
the  mitral-valve  factor  be  best  made  out  to  differentiate  it  from 
the  other  valves?  The  tricuspid- valve  factor?  The  aortic-semi- 
lunar-valve  factor  ?  The  pulmonary-semilunar- valve  factor  ?  Ex- 
plain why  you  listen  in  certain  regions  for  the  different  valve 
sounds. 

While  a  cardiogram  is  being  made,  listen  to  the  heart  sounds. 
Compare  the  sounds,  in  point  of  time,  with  the  features  of  the  car- 
diogram tracing. 

While  a  sphygmogram  is  being  taken  listen  to  the  heart  sounds. 

XXIII.  THE  VASOMOTOR  MECHANISM. 

1.  The  General  Controlling  Centre  in  the  Medulla. — Anaes- 
thetize a  large  frog  lightly  with  ether.  Cut  both  vagus  nerves,  in 
order  to  exclude  changes  in  the  heart-beat,  through  inhibition  or 
augmentation,  from  the  result.  Carefully  avoiding  hemorrhage, 
expose  the  brain  and  upper  part  of  the  cord. 

[no] 


CIRCULATION  OF  THE  BLOOD. 

A  good  index  of  general  vasomotor  changes  is  the  speed  of 
blood-flow  through  the  capillary  vessels  of  the  frog's  web.  If  the 
arterioles  supplying  the  capillaries  are  constricted,  the  speed  of 
flow  through  the  capillaries  will  be  diminished.  If  the  arterioles 
are  dilated,  the  speed  of  flow  through  the  capillaries  will  be  in- 
creased. 

Keeping  the  brain  and  cord  moistened  with  physiological  salt 
solution,  arrange  the  web  under  a  microscope  for  observation  with 
a  medium  high  power.  Note  the  size  of  the  larger  vessels,  the  ar- 
terioles and  venules,  and  the  speed  of  flow  through  the  capillaries. 
The  diameter  of  one  of  the  arterioles  may  be  measured  by  means 
of  the  micrometer  eyepiece. 

Excise  the  cerebral  hemispheres  and  optic  lobes,  controlling 
hemorrhage,  if  necessary,  by  packing.  Allow  five  or  ten  minutes  for 
the  frog  to  recover  from  the  shock  of  the  operation.  Now  examine 
the  web  again.  Is  there  any  observable  change  in  the  size  of  the 
arterioles  or  in  the  speed  of  blood-flow  through  the  capillaries? 
Has  there  been  any  marked  interference  with  the  tone  of  the  blood- 
vessels? What  conclusion  can  you  draw  concerning  the  presence 
of  a  vasomotor  centre  in  the  parts  excised  ? 

Observe  the  web  for  some  time.  Are  there  any  rhythmical 
changes  in  the  diameters  of  the  larger  vessels  discernible  ? 

2.  Inject  a  drop  of  saturated  curare  solution  under  the  skin  of 
the  frog  in  order  just  to  paralyze  the  motor  nerves  without  affect- 
ing the  innervation  of  the  blood-vessels.    This  is  done  to  keep  the 
frog  quiet  during  the  ensuing  stimulations.    Set  up  an  inductorium 
arranged  for  weak  tetanizing  currents. 

While  observing  the  flow  through  the  web,  stimulate  the  medulla 
with  fine  needle  electrodes.  What  is  the  effect  on  the  blood-flow 
through  the  web  ?  Is  this  a  constrictor  or  dilator  effect  ? 

3.  Sever  the  medulla  from  the  cord  by  a  clean  transverse  cut 
with  a  sharp  scalpel.     Again  observe  the  flow  through  the  web. 
Is  the  speed  increased  or  decreased?    Is  there  a  constriction  or 
dilatation  of  the  arterioles?    What  is  the  function  of  the  centre  in 
the  medulla  ?    Is  its  action  constant  or  intermittent  ? 

[mi 


LABORATORY  MANUAL  OF  PHYSIOLOGY. 

4.  While  observing  the  capillary  flow,  stimulate  the  cut  end  of 
the  cord  with  a  weak  tetanizing  current.    What  is  the  effect  on  the 
calibre  of  the  blood-vessels  ?    What  conclusion  can  you  draw  con- 
cerning the  function  of  the  cord  as  a  conductor  of  vasomotor  im- 
pulses ? 

5.  Cord  Centres. — Allow  the  frog  to  rest  for  ten  or  fifteen  min- 
utes, making  an  observation  of  the  web  at  frequent  intervals.    Is 
there  any  diminution  in  speed  of  flow?    Is  there  any  resumption 
of  vasomotor  tone?    If  so,  what  conclusion  can  you  draw  con- 
cerning the  cord  as  a  vasomotor  centre  ? 

6.  Destruction  of  the  Cord. — Expose  the  heart  and  vessels 
of  the  mesentery.    Note  the  complete  filling  of  the  heart  at  each 
diastole  and  the  size  of  the  mesenteric  vessels.     Now  insert  a 
seeker  into  the  spinal  canal,  destroying  the  cord.     What  is  the 
effect  on  the  filling  of  the  heart  and  the  abdominal  vessels  ?    What 
disturbance  of  the  vasomotor  mechanism  has  occurred  through 
the  destruction  of  the  cord  ? 

Examine  the  web.  How  does  it  compare  with  the  condition 
which  prevailed  before  the  destruction  of  the  cord  ? 

7.  Vasomotor  Centres  Outside  of  the  Cord. — Pith  another 
frog,  but  do  not  destroy  the  cord.    Wait  five  minutes  for  the  vaso- 
motor tone  to  be  re-established.    Examine  the  web  and  note  the 
size  of  the  vessels  and  speed  of  flow. 

Destroy  the  cord  in  the  same  way  as  before  and  note  the  change 
in  the  web  flow.  Observe  the  web  at  short  intervals,  for  fifteen  to 
twenty  minutes,  or  longer  if  necessary.  Is  there  any  return  of 
vascular  tonus?  Explain. 

8.  Vasomotor  Reflexes. — Lightly  curarize  a  frog.    Carefully 
isolate  the  sciatic  nerve  of  one  thigh.     Examine  the  blood-flow 
through  the  web  of  the  opposite  foot.    Cut  the  nerve.    Is  there  any 
effect  on  the  capillary  flow  through  the  web  ?    Is  this  effect  con- 
strictor or  dilator? 

After  five  minutes,  stimulate  the  central  end  of  the  cut  nerve  with 
a  weak  tetanizing  current.  What  is  the  result  on  the  circulation 
through  the  web  ? 

[112]  . 


CIRCULATION  OF  THE  BLOOD. 

Examine  the  web  of  the  foot  on  the  same  side  as  the  cut  nerve. 
Stimulate  the  distal  end  of  the  cut  nerve.  What  is  the  effect  on  the 
capillary  circulation  ?  Explain. 

Prepare  another  frog,  cocainizing  the  sciatic  nerve  before  cut- 
ting. Compare  the  web  circulation  in  this  case,  before  and  after 
cutting,  with  the  result  obtained  when  the  nerve  was  not  cocainized. 

XXIV.  RECORD  OF  THE  BLOOD  PRESSURE  IN  THE  RABBIT. 

1.  Preparation. — Under  the  skin  of  the  rabbit  inject  £  grain 
of  morphine  sulphate.  Anaesthetize  lightly  with  ether.  Expose 
both  carotid  arteries,  and  isolate  and  identify  the  nerves  running 
with  them.  Pass  two  loops  of  thread  about  the  vagus  and  de- 
pressor nerves  of  each  side.  Introduce  a  cannula  into  the  trachea. 
Clean  the  carotid  of  one  side  for  a  distance  of  three  or  four  centi- 
metres. Pass  two  loops  of  thread  around  the  artery.  Tie  the 
upper  ligature  as  far  away  from  the  heart  as  possible.  Clamp  the 
artery  near  the  lower  part  of  its  isolation.  With  a  pair  of  fine- 
pointed  scissors  make  a  V-shaped  opening  in  the  vessel  near  the 
upper  ligature,  using  the  tied  ligature  to  manipulate  the  artery. 
Introduce  a  fine  blunt-pointed  seeker  into  the  lumen  of  the  cut 
vessel  toward  the  heart.  Using  this  seeker  as  a  guide,  insert  into 
the  artery  a  glass  cannula  made  for  the  purpose.  Tie  this  securely 
in  the  vessel  by  means  of  the  remaining  untied  ligature. 

In  the  mean  time  the  apparatus  for  transmission  and  recording 
of  the  blood  pressure  should  have  been  made  ready.  This  consists 
of  a  revolving  drum,  covered  with  smoked  glazed  paper;  a  mer- 
cury manometer  with  an  upright  and  writing  style  resting  on  the 
mercury  of  one  limb  of  the  manometer  and  arranged  to  write  on 
the  drum;  the  other  limb  of  the  manometer  is  connected  through 
pressure  tubing  with  the  cannula  which  has  been  introduced  into 
the  carotid. 

The  proximal  limb  of  the  manometer,  and  the  tubing  connected 
with  it,  are  filled  from  a  pressure  bottle,  with  a  half-saturated  solu- 
tion of  sodium  carbonate  or  some  other  salt  solution  to  prevent 
clotting  of  the  blood  which  may  find  its  way  into  the  tubing. 

8  ["3] 


LABORATORY  MANUAL  OF  PHYSIOLOGY. 

The  manometer  tubing  is  clamped  off  and  the  pressure  in  the 
manometer  and  connections  is  raised  to  approximate  the  estimated 
arterial  pressure  of  the  rabbit. 

The  artery  cannula  is  then  filled  with  o.8-per-cent  solution  of 
NaCl,  care  being  taken  to  get  rid  of  all  air  bubbles  both  in  the 
cannula  and  manometer  connections.  The  manometer  tube  is  then 
connected  with  the  arterial  cannula,  the  clamp  is  removed  from  the 
tubing  and  from  the  artery  at  the  same  time,  and  the  pressure  in 
the  artery  is  transmitted  through  the  tubing  to  the  mercury  in  the 
proximal  limb  of  the  manometer  which  falls  and  rises  in  the  distal 
limb. 

Before  the  mercury  in  the  manometer  was  put  under  extra  press- 
ure, a  base  line  should  have  been  drawn  around  the  drum  to  in- 
dicate the  atmospheric  pressure.  The  height  of  the  tracing  above 
this  base  line,  multiplied  by  2,  gives  the  pressure  in  terms  of 
mercury. 

2.  Note  the  rise  and  fall  of  pressure  synchronous  with  the  heart- 
beat.   Where  the  beat  is  rapid,  as  it  is  normally  in  the  rabbit,  the 
excursion  of  the  manometer  style  is  not  an  accurate  index  of  the 
variations  of  pressure.    The  inertia  of  the  mercury  is  too  great  to 
follow  exactly  small  and  rapid  variations  in  pressure. 

Note  the  larger  waves  in  the  tracing.  These  are  due  to  changes 
in  arterial  pressure  brought  about  by  inspiration  and  expiration. 
Observe  the  respiratory  movements  and  compare  them  with  the 
respiratory  waves  of  the  tracing.  Is  there  a  rise  or  fall  of  blood 
pressure  with  inspiration?  with  expiration?  Does  the  change 
correspond  exactly  to  the  respiratory  movement? 

Are  there  any  other  pressure  waves  in  the  tracing  aside  from  the 
pulse  waves  and  the  respiratory  waves  ?  Explain. 

3.  Effect  of  Vagus  Stimulation. — While  the  tracing  is  being 
taken,  tie  one  vagus  with  two  ligatures  and  cut  between.    Note  any 
change  in  the  pressure  tracing.    Mark  on  the  tracing  the  place  at 
which  the  nerve  was  tied  and  cut. 

(a)  Stimulate  the  peripheral  end  of  the  cut  nerve  with  a  weak 
tetanizing  current.  Is  there  any  effect  on  blood  pressure  or  upon 

t"4] 


CIRCULATION  OF  THE  BLOOD. 

the  rate  and  strength  of  the  heart-beat  ?  Stimulate,  again,  with  a 
somewhat  stronger  current  and  note  the  effect. 

Increase  the  strength  of  the  current,  a  little  each  time,  until  the 
full  inhibitory  vagus  effect  is  obtained.  What  is  the  result  on  blood 
pressure  ?  What  is  the  after-effect  of  vagus  stimulation  ? 

(b)  Stimulate  the  central  end  of  the  divided  nerve  with  medium 
strong  tetanizing  current.  What  is  the  effect  on  blood  pressure? 
Note  the  respiratory  movements  before  and  during  the  stimula- 
tion of  the  central  end  of  the  nerve. 

4.  Depressor  Stimulation. — Tie  the  two  ligatures  which  were 
passed  around  this  nerve  and  cut  the  nerve  between  the  ligatures. 

(a)  Stimulate  the  peripheral  end  of   the  cut  nerve;  (the   end 
toward  the  heart)  with  medium   strong  tetanizing  currents.     Is 
there  any  effect  on  the  rate  or  strength  of  the  heart-beat  or  upon 
the  blood  pressure? 

(b)  Stimulate  the  central  end  of  the  divided  nerve  in  the  same 
way.    Is  there  any  effect  on  the  rate  and  strength  of  the  heart-beat 
or  upon  the  blood  pressure,  or  both  ?    How  does  this  tracing  com- 
pare with  that  obtained  through  stimulation  of  the  peripheral  end 
of  the  cut  vagus,  using  the  same  strength  of  stimulus  ? 

Compare  the  result  obtained  through  stimulating  the  central 
end  of  the  depressor  with  that  obtained  through  stimulating  the 
peripheral  end.  Is  the  depressor  effect  a  direct  or  an  indirect 
effect?  Is  the  nerve  an  efferent  or  an  afferent  pathway?  If  it 
forms  part  of  a  reflex  arc,  what  nerve  pathways  form  the  other 
limb  or  limbs  of  the  arc  ? 

(c)  Now  cut  the  intact  vagus  of  the  opposite  side.    Note  any 
further  change  in  the  blood-pressure  tracing. 

Again  stimulate  the  central  end  of  the  depressor  nerve.  Is 
there  any  fall  in  blood  pressure  ?  Is  there  any  slowing  of  the  heart- 
beat ?  What  change  has  vagus  section  made  in  the  depressor  re- 
flexes ?  Explain.  In  the  light  of  the  above  observations,  what  are 
the  functions  of  the  depressor  nerve  ? 

5.  Section  of  the  Cervical  Sympathetic. — Using  the  same 
rabbit,  observe  the  appearance  of  the  blood-vessels  in  the  two  ears. 


LABORATORY  MANUAL  OF  PHYSIOLOGY. 

Secure  the  cervical  sympathetic  nerve  on  one  side  with  two  liga- 
tures. Cut  the  nerve  between  the  ligatures.  After  a  short  time, 
again  compare  the  two  ears.  Is  there  any  change  in  vascularity  of 
the  ear  of  the  side  upon  which  the  nerve  was  cut  as  compared  with 
the  ear  of  the  sound  side  ? 

While  observing  the  two  ears,  stimulate  the  peripheral  end  of 
the  cut  nerve.  What  change  occurs  in  the  blood  supply  to  the  ear 
of  the  stimulated  side?  Note  any  other  phenomena  which  occur 
during  stimulation  of  the  cervical  sympathetic  nerve.  What  is  the 
function  of  the  nerve  as  far  as  the  vascular  supply  to  the  ear  is  con- 
cerned ?  How  does  the  temperature  of  the  ear  on  the  operated  side 
compare  with  that  of  the  ear  on  the  sound  side  ? 

Locate  the  cardiac  impulse  on  the  chest  wall  of  the  rabbit  and 
introduce  into  the  heart  a  fine  knitting-needle.  This  will  move 
with  the  heart-beat  and  serve  as  an  indicator.  Place  a  bit  of  ab- 
sorbent cotton  saturated  with  chloroform  over  the  end  of  the  tra- 
cheal  cannula.  Note  the  effect  on  the  heart-beat.  Kill  the  animal 
writh  chloroform.  Note  which  stops  first,  heart-beat  or  respiration. 
After  the  respirations  and  excursions  of  the  heart  needle  have  both 
stopped,  open  the  thorax  and  observe  whether  or  not  the  heart  is 
still  feebly  beating. 

6.  Effect  of  Hemorrhage  on  Blood  Pressure. — Prepare  a 
rabbit,  as  before,  for  a  blood-pressure  record.  In  addition  intro- 
duce a  long  glass  cannula  into  the  opposite  carotid  artery  and 
prepare  the  jugular  vein  for  transfusion. 

Take  a  normal  blood-pressure  record  for  comparison.  While 
this  is  being  taken,  remove  the  clamp  from  the  opposite  carotid 
until  50  c.c.  of  blood  have  been  shed.  Note  any  change  in  blood 
pressure  during  the  bleeding.  Shut  off  the  artery  after  the  loss  of 
the  50  c.c.  of  blood.  If  the  pressure  has  fallen  during  the  hemor- 
rhage, does  it  continue  low  or  does  it  recover  after  the  bleeding 
has  ceased?  Explain. 

Bleed  again  another  50  c.c.  of  blood.  Is  there  any  further  fall 
in  pressure  ?  If  so,  does  it  again  rise  after  the  cessation  of  the  hem- 
orrhage ? 

[116] 


CIRCULATION  OF  THE  BLOOD. 

Bleed  again,  if  necessary,  until  the  blood  pressure  ceases  to  re- 
cover. Now  introduce  into  the  jugular  vein,  slowly,  o.8-per-cent 
NaCl  solution,  until  50  or  100  c.c.  have  been  transfused.  Does  the 
pressure  again  rise  ?  If  not,  continue  the  perfusion. 

Perfuse  four  or  five  times  as  much  of  the  salt  solution  as  there 
was  blood  lost.  Is  there  any  noticeable  rise  of  pressure  above  the 
normal  ?  If  not,  explain 

7.  Shock  and  Blood  Pressure. — Prepare  another  rabbit  for 
blood-pressure  recording,  isolating  the  vagus  and  depressor  nerves 
at  the  same  time.  Take  a  normal  blood-pressure  tracing.  While 
this  is  being  done,  stimulate  the  peripheral  end  of  the  vagus. 
Take  another  tracing,  stimulating  the  depressor  nerve.  These 
tracings  will  serve  as  checks  to  the  results  obtained  later. 

While  the  pressure  tracing  is  being  taken,  open  the  abdomen  by 
a  long  median  incision.  Expose  and  handle  the  abdominal  viscera. 
What  is  the  effect  on  the  blood  pressure  ? 

If  the  pressure  has  fallen,  stimulate  the  depressor  nerve.  Does 
the  pressure  fall  still  more  ? 

While  the  pressure  is  low,  inject  into  the  jugular  vein  2  c.c.  of  a 
i  to  10,000  solution  of  adrenalin  chlorid.  What  is  the  effect  on 
blood  pressure  and  rate  of  the  heart-beat  ? 

The  adrenalin  effect  will  disappear  after  a  short  time.  Cut  both 
vagus  nerves  and  repeat  the  adrenalin  injection.  Is  there  any  dif- 
ference in  effect  on  the  rate  of  the  heart-beat  as  compared  with  the 
first  result  obtained  ? 

XXV.  PERFUSION  OF  THE  RABBIT'S  HEART. 

Bleed  the  rabbit  of  experiment  7  from  both  carotids,  and  defi- 
brinate  the  blood  as  shed.  Mix  this  with  equal  parts  of  o.8-per- 
cent  NaCl  solution  which  has  been  warmed  to  37°  C. 

Open  the  thorax.  Excise  the  heart  which  will  probably  con- 
tinue to  beat.  Introduce  and  tie  a  small  cannula  into  the  opening  of 
one  coronary  artery  where  it  leaves  the  aorta.  Free  the  cannula  of 
air  by  filling  it  with  physiological  saline.  Place  the  defibrinated 
blood  mixture  in  a  bottle  with  a  side  piece  and  resting  in  a  water- 


LABORATORY  MANUAL  OF  PHYSIOLOGY. 

bath  kept  at  a  constant  temperature  of  38°  C.  Raise  this  per- 
fusion  bottle  above  the  heart  to  give  a  pressure  of  one  hundred 
millimetres  of  mercury. 

Connect  the  perfusion  bottle  with  coronary  cannula,  place  the' 
heart  in  a  normal  salt-solution  bath,  and  begin  perfusion.  Note  the 
strength  and  rhythm  of  the  heart-beat  as  well  as  the  rate.  Is  the 
mammalian  heart  dependent  upon  the  central  nervous  system  for 
the  origination  of  its  beat  or  for  the  co-ordination  of  the  beat  ? 

After  twenty  minutes,  if  the  heart  is  still  beating  strongly,  dis- 
connect from  the  defibrinated-blood  perfusion,  and  perfuse, 
instead,  with  warm  o.8-per-cent  NaCl  solution.  How  does  this 
solution  compare  with  the  blood  mixture  in  maintaining  the 
beat  of  the  heart  ? 

XXVI.  EFFECT  OF  TEMPERATURE  ON  THE  HEART-BEAT. 

Narcotize  and  etherize  a  medium-sized  rabbit.  Place  on  rabbit- 
board,  back  down.  Expose  both  carotid  arteries,  in  the  neck  re- 
gion. Pass  under  both  arteries  a  large  glass  tube  connected  with 
a  pressure  bottle.  Insert  in  the  heart,  through  the  thorax,  a  knit 
ting-needle  indicator.  Count  the  heart-beats  per  minute.  Also, 
count  the  respirations.  Now  allow  water,  heated  to  40°  C.,  t^ 
flow  through  the  tube  running  under  the  arteries.  Note  the 
increase  in  the  rate  of  the  heart-beat  and  in  the  frequency  of 
respiration. 

Stop  the  flow  of  the  hot  water  through  the  tube.  Allow  the  heart- 
beat and  respiratory  rate  to  return  to  normal  and  then  run  ice 
water  through  the  tube.  What  is  the  effect  of  the  cold  as  compared 
with  the  application  of  heat  to  the  circulating  blood  ? 

In  all  of  the  experiments  on  the  rabbit  where  it  has  not  been 
specified,  the  animal  should  be  narcotized  with  morphine  and  an- 
aesthetized with  ether. 

"XXVII.  ESTIMATION  OF  HUMAN  BLOOD  PRESSURE. 

A  number  of  instruments  have  been  devised  for  estimating  blood 
pressure  in  man.  It  is  obviously  impracticable  to  determine  the 

[118] 


CIRCULATION  OF  THE  BLOOD. 

blood  pressure  in  the  same  way  that  was  done  in  the  case  of  the 
rabbit. 

The  Riva-Rocci  sphygmomanometer  or  some  modification  of  it 
is  in  most  general  use,  and,  although  not  the  most  accurate  form  of 
apparatus,  perhaps,  for  the  purpose,  it  certainly  is  most  convenient. 

The  apparatus  consists  of  an  elastic  tube,  blind  at  both  ends  and 
having  a  side  piece  for  connection  with  the  air-inflating  pump  (see 
Fig.  35).  This  tube  is  covered  by  a  leather  cuff,  so  that  when  it  is 


FIG.  35.— Sphygmomanometer  (Schematic).    (Description  in  text.) 

adjusted  around  the  arm  or  forearm  the  pressure  is  exerted  in  the 
direction  of  the  limb  (see  Fig.  35,  A  and  C).  The  arm  tube  is  con- 
nected with  some  form  of  air  pump  (Fig.  35,  P)  and  with  a  mercury 
manometer  (Fig.  35,  B).  By  inflating  the  tube  until  sufficient 
pressure  upon  the  arm  is  produced  to  shut  off  the  pulse  at  the  wrist, 
a  rough  estimate  of  the  systolic  blood  pressure  is  obtained.  This 

["9] 


LABORATORY  MANUAL  OF  PHYSIOLOGY. 

pressure  is  read  off  on  the  manometer  scale  in  millimetres  of  mer- 
cury. 

If  now  the  pressure  is  released  until  the  widest  oscillations  of 
the  mercury  meniscus  are  just  secured  and  a  reading  is  then  taken, 
the  diastolic  pressure  is  shown.  This  may  be  more  accurately  de- 
termined by  palpating  the  pulse  and  inflating  the  tube  until  the 
first  perceptible  diminution  of  the  pulse  occurs.  To  reduce  error 
as  much  as  possible,  the  arm  tube  and  cuff  should  be  at  least 
twelve  centimetres  in  width. 

With  some  such  instrument  as  that  described  above,  make  a 
number  of  determinations  of  both  systolic  and  diastolic  pressure 
of  one  individual.  Repeat  the  observations  on  other  individuals  of 
the  class.  If  there  are  other  instruments  at  hand  for  this  same 
purpose,  make  observations  with  several  different  instruments  on 
the  same  individual. 

Make  an  observation  on  a  normal  individual.  Allow  the  subject 
to  take  several  whiffs  of  amyl  nitrite  and  observe  the  blood  pressure. 


120] 


CHAPTER  VI. 
SECRETION— DIGESTION— ABSORPTION. 

I.  SECRETION  OF  SALIVA. 

Mechanism  of  Secretion. — Demonstration. — Secretion  may 
be  divided  into  two  stages — the  productive  stage,  during  which  the 
secretory  products  are  being  formed  in  the  gland  cells,  and  the 
eliminative  stage,  during  which  the  products  already  formed  pass 
out  of  the  cells  and  through  the  gland  ducts  to  the  surface  where  the 
secretion  performs  its  particular  function. 

The  elimination  of  the  secretion  may  be  brought  about  either 
by  a  nervous  stimulus  of  the  gland  cells  or  a  chemical  stimulus,  or 
both.  The  mechanism  of  the  elimination  of  the  salivary  secretions 
furnishes  an  excellent  example  of  the  influence  of  nervous  im- 
pulses. This  is  not  of  so  much  importance  in  itself,  as  it  is  in  serv- 
ing as  a  type  of  the  nervous  mechanism  of  secretions  in  general. 

Of  the  salivary  glands,  the  one  which  has  been  chiefly  studied 
and  used  to  demonstrate  this  mechanism  is  the  submaxillary 
gland. 

Place  a  dog  under  ether  and  tie  to  the  dog-board.  Clip  the  hair 
from  the  jaws  and  neck  and  shave  the  skin.  Make  an  incision 
through  the  skin  of  the  lower  jaw,  along  its  inner  border,  beginning 
just  in  front  of  the  insertion  of  the  anterior  belly  of  the  digastric 
muscle  and  extending  backward  through  the  platysma  muscle  to 
the  transverse  process  of  the  first  cervical  vertebra. 

Expose  the  jugular  vein  and  its,  branches,  including  those  which 
drain  the  submaxillary  gland.  Tie  all  venous  branches  which  pass 
below  and  in  front  of  the  gland,  excepting  those  which  come  from 
the  gland  itself.  The  veins  should  be  tied  between  two  ligatures, 

[121] 


LABORATORY  MANUAL  OF  PHYSIOLOGY. 

and  the  intervening  portion  excised.  Clean  the  masseter  and  di- 
gastric muscles  of  cellular  tissue.  Avoid  irjury  to  the  facial  artery 
and  the  gland  duct  which  lies  between  it  and  the  masseter  muscle. 
Carefully  separate  the  digastric  muscle  from  the  artery,  and  tie  the 
branch  which  supplies  the  muscle.  Divide  the  digastric  and  mylo- 
hyoid  muscles  and,  being  careful  to  avoid  injury  to  the  structures 
beneath,  turn  the  muscle  flaps  back. 

The  submaxillary  gland  should  also  be  gently  drawn  upward 
and  backward.  The  following  structures  will  now  be  exposed  to 
view: 

In  front  of  the  posterior  insertion  of  the  digastric  and  in  front  of 
and  below  the  reflected  gland,  the  carotid  artery  is  seen.  Crossing 
this  is  the  hypoglossal  nerve,  H  (see  Fig.  36),  and  running  along 


FIG.  36.— Dissection  of  Submaxillary  Region,  Dog.  (After  Bernard.)  G,  Sub- 
maxillary  gland  ;  /,  jugular  vein  ;  C,  carotid  artery ;  //,  hypoglossal  nerve ;  L,  lingual 
nerve ;  T,  chorda  tympani ;  £>,  ducts  of  submaxillary  and  sublingual  glands  (S)  ; 
Dg;  digastric  muscle ;  M,  mylo-hyoid  muscle. 

with  the  artery  are  filaments  of  the  sympathetic  nerves.  Entering 
into  and  passing  out  of  the  hilum  of  the  gland  are  seen  the  chorda 
tympani  nerve  branch  to  the  gland,  the  branch  of  the  facial  artery 
to  the  gland,  and  the  gland  duct. 

Beneath  the  reflected  mylo-hyoid  muscle  is  seen  the  lingual 
nerve  (Fig.  36,  L).  Trace  this  nerve  to  the  ramus  of  the  jaw.  At 
this  point  a  small  branch  will  be  exposed  which,  in  close  proximity 

[122] 


SECRETION— DIGESTION— ABSORPTION. 

to  the  duct,  runs  backward  to  the  gland.  This  is  the  chorda  tym- 
pani  (Fig.  36,  T). 

Pass  a  thread  under  the  chorda  to  use  later  in  handling  the 
nerve  for  stimulation.  Divide  the  hypoglossal  nerve  and  expose 
the  sympathetic  filaments.  Pass  a  thread  around  these  also.  Iden- 
tify the  submaxillary  duct  and  introduce  and  tie  a  cannula  into  it. 
The  cannula  should  end  in  a  small  rubber  tube.  This  is  closed 
by  an  artery  clip  until  it  is  desired  to  collect  the  secretion.  The 
introduction  of  the  cannula  may  be  facilitated  by  first  stimulat- 
ing the  chorda  for  a  short  time  with  a  weak  tetanizing  current, 
thus  distending  the  duct  with  secretion. 

Small  graduated  glass  cylinders  are  provided  for  collecting  the 
secretion  from  the  cannula  in  the  gland  duct.  These  may  be 
changed  at  any  desired  interval  of  time,  say  every  five  or  ten 
minutes.  The  rate  of  flow  is  determined  by  the  amount  of 
secretion  eliminated  in  a  given  time  period. 

1.  Observe  the  rate  of  flow  from  the  gland  before  stimulation. 
Has  the  anaesthetic  any  stimulating  influence  on  the  salivary  flow  ? 
This  observation  should  cover  a  period  of  five  minutes. 

2.  Stimulate  the  chorda  with  a  weak  tetanizing  current.    How 
is  the  rate  of  salivary  flow  affected  ?    Compare  the  appearance  of 
the  blood-vessels  of  the  gland  during  stimulation  of  the  nerve  with 
the  vascular  condition  before  stimulation. 

3.  Allow  the  preparation  to  rest  for  several  minutes.    Stimulate 
the  sympathetic.    Is  there  any  marked  effect  on  the  rate  of  flow  ? 
What  is  the  effect  upon  the  condition  of  the  blood  supply  to  the 
gland  ? 

4.  Paint  the  submaxillary  ganglion  with  a  o.i-per-cent  solution 
of  nicotine.    Nicotine,  in  weak  solution,  paralyzes  nerve  cells,  but 
not  nerve  fibres.    Stimulate  the  chorda  again.    Is  there  still  an  ac- 
celerator effect  on  the  salivary  secretion?    Are  the  fibres  of  the 
chorda  broken  by  nerve  cells  in  this  ganglion  ? 

Now  paint  the  chorda  with  nicotine  where  it  enters  the  hilum  of 
the  gland.  Stimulate  the  chorda  again.  Is  there  any  effect  on  the 
flow  of  the  secretion  ?  Stimulate  at  the  hilum  itself.  Is  there  any 


LABORATORY  MANUAL  OF  PHYSIOLOGY. 

effect  on  the  flow  of  secretion  ?  Does  the  chorda  run  directly  to  the 
gland  cells  without  interruption?  If  not,  where  is  the  point  of 
transfer  ? 

5.  Wash  the  gland  thoroughly  with  warm  physiological  saline. 
After  a  time  the  nicotine  effect  will  wear  off.  Isolate  and  intro- 
duce a  cannula  into  the  femoral  vein,  centrally.  Inject  into  the 
vein  J  grain  atropine  sulphate.  Note  the  effect  on  the  flow  of  sa- 
liva. Stimulate  the  chorda.  Is  there  an  increased  flow  of  the 
secretion?  Empty  the  duct  of  secretion.  With  a  hypodermic 
syringe  inject  into  the  duct  a  few  drops  of  a  2-per-cent  solution  of 
pilocarpine  nitrate.  Stimulate  the  chorda  again.  Is  any  effect  now 
obtainable  on  the  salivary  flow  ?  After  several  minutes  stimulate 
again.  The  atropine  effect  has  once  more  asserted  itself. 

Does  stimulation  of  the  nerve  still  cause  a  dilatation  of  the  gland 
vessels?  Is  the  effect  of  the  nerve  stimulation  in  causing  an  in- 
crease in  flow  of  secretion  purely  a  vaso-dilator  effect  or  is  it  due  to 
a  stimulation  of  the  gland  cells  themselves  ?  Explain. 

II.  To  SHOW  CHANGES  IN  THE  GLAND  CELLS  FOLLOWING 
CHORDA  STIMULATION. 

Make  another  preparation  of  submaxillary  gland,  duct,  and 
nerve.  Stimulate  the  nerve  with  a  weak  tetanizing  current  until 
the  flow  of  the  secretion  ceases.  Allow  an  interval  of  five  minutes' 
rest  and  repeat  the  stimulation.  Continue  to  complete  exhaustion 
of  the  gland.  Remove  both  submaxillary  glands.  Cut  out  small 
portions  of  each  and  make  frozen  sections  of  the  fresh  glands. 
Mount  in  normal  salt  solution  or  in  glycerin.  Examine  sections 
from  the  two  glands  under  the  microscope.  Compare  the  appear- 
ance of  the  cells  of  the  stimulated  gland  with  that  of  the  resting 
gland. 

Harden  the  remainder  of  the  two  glands  in  absolute  alcohol; 
embed  in  paraffin;  cut  sections;  and  stain  with  carmine.  Com- 
pare the  stained  sections  of  the  two  glands  with  each  other  and 
with  the  frozen  sections  of  the  fresh  glands. 


SECRETION— DIGESTION— ABSORPTION. 

III.  SALIVARY  DIGESTION. 

1.  Chemical  Constituents  of  Saliva. — Chew  a  piece  of  par- 
affin gum  or  inhale  a  little  ether  vapor.    The  flow  of  saliva  is 
thus  stimulated.     Collect  the  secretion  in  a  clean  porcelain  cap- 
sule.   Filter  and  divide  the  nitrate  into  five  portions. 

(a)  Test  the  first  portion  for  its  reaction  with  litmus  paper. 
Is  it  alkaline  or  acid  ?    Is  the  reaction  very  decided  in  either  di- 
rection ? 

(b)  To  the  second  portion  add  dilute  acetic  acid.    The  pres- 
ence of  mucin  is  indicated  by  the  formation  of  a  precipitate. 

(c)  To  a  third  portion  add  a  few  drops  of  a  silver-nitrate  solu- 
tion.   A  precipitate  of  silver  chlorid  which  is  soluble  in  ammonia 
and    insoluble    in  nitric  acid  is  indicative  of  the  presence  of 
chlorids. 

(d)  To  another  portion  add  dilute  acetic  acid  and  filter.    Test 
the  filtrate  with  Millon's  reagent.     The  presence  of  proteids  is 
shown  by  the  production  of  a  red  coloration  or  precipitate. 

2.  Action  on  Starches. — (a)  To  some  boiled  starch  paste  add 
a  few  drops  of  iodine.    A  blue  coloration  will  occur.    To  some 
powdered  starch  add  a  few  drops  of  iodine.    A  blue  color  test  will 
also  be  obtained.     Both  cooked  and  raw  starch  respond  to  the 
iodine  test. 

(b)  To  a  test-tube  partly  filled  with  a  dilute  Fehling's  solution 
add  a  little  of  the  starch  paste.    Boil  the  mixture.    There  should 
be  no  reduction  of  the  copper  sulphate  of  the  Fehling's  solution. 
The  copper  salt  is  reduced  by  any  of  the  reducing  sugars. 

(c)  To  another  portion  of  Fehling's  solution  add  a  few  drops 
of  a  dilute  solution  of  dextrose.    Heat  to  boiling  and  note  the  for- 
mation of  a  copious  precipitate,  first  yellow,  and,  as  the  heating  is 
continued,  changing  to  a  reddish  color.    This  is  the  cuprous  and 
later  cupric  oxide  formed  by  the  reduction  of  the  copper  salt  in  the 
test  solution. 

(d)  Repeat  the  test  with  maltose  instead  of  dextrose.    Is  re- 
duction obtained? 


LABORATORY  MANUAL  OF  PHYSIOLOGY. 

(e)  To  another  portion  of  the  test  solution  add  lactose.    Is  the 
copper  salt  reduced  ? 

(/)  Test  a  solution  of  cane  sugar  for  reduction.    Result  ? 

(g)  Partly  fill  three  test-tubes  with  starch  paste.  Mark  them 
I,  II,  and  III.  To  I  add  some  of  the  filtered  saliva.  To  II  add 
some  saliva  that  has  been  heated  to  boiling.  To  III  add  some 
saliva  that  has  been  neutralized  with  HC1  and  has  had  enough 
acid  added  to  bring  the  acidity  up  to  i  per  cent  HC1.  Place  the 
three  tubes  in  a  water-bath  or  in  the  incubator  kept  at  a  tempera- 
ture of  38°  C.  In  five  minutes  remove  the  tubes  and  test  the 
three  for  reducing  sugar  with  Fehling's  solution.  Is  there  any 
reducing  sugar  in  I  ?  in  II  ?  in  HI  ?  Explain. 

(//)  Make  a  solution  of  dextrin.  To  this  solution  add  a  few 
drops  of  iodine.  Note  the  wine-color  reaction. 

(t)  To  a  few  cubic  centimetres  of  the  dextrin  solution  add  an 
equal  quantity  of  saliva.  Place  in  the  water-bath  or  incubator  for 
one  hour  at  a  temperature  of  38°  C.  At  the  end  of  this  time  test  the 
solution  with  iodine  for  the  red  dextrin  reaction.  Is  there  any 
color  reaction?  Test  for  reducing  sugar. 

(f)  Mix  equal  quantities  of  boiled  starch  and  saliva  in  a  test- 
tube.    Place  in  the  warm  water-bath.    Place  several  drops  of  dilute 
iodine  solution  on  a  white  porcelain  slab.    At  five-minute  intervals, 
by  means  of  a  glass  stirring-rod,  add  a  drop  of  the  digesting  starch 
to  a  drop  of  the  iodine  on  the  slab.    Continue  this  until  a  color  re- 
action is  no  longer  obtained.    \Yhat  changes  occur  in  the  color 
reaction  as  digestion  progresses  ? 

(k)  Take  some  boiled  starch  into  the  mouth  and  go  through  the 
movements  of  mastication.  At  the  end  of  one  minute  spit  this  out 
into  some  boiling  water  in  a  beaker.  Test  a  portion  of  the  mixture 
for  sugar,  and  another  portion  for  starch .  Repeat  with  another  por- 
tion of  starch,  keeping  it  in  the  mouth  two  minutes.  Chew  another 
portion  for  five  minutes  and  test  again  for  starch  and  sugar.  Does 
the  lest  for  starch  diminish  in  intensity,  and  the  test  for  sugar 
increase  ? 

(/)  Mix  a  portion  of  fibrin  in  a  test-tube  with  some  saliva. 

[126] 


SECRETION—  DIGESTION—  ABSORPTION. 

Place  in  the  incubator  for  twenty  minutes:  At  the  end  of  this  time 
remove  and  test  the  mixture  for  peptones.  Has  any  of  the  proteid 
been  digested  ? 

(;;z)  Add  some  saliva  to  a  starch  solution  in  a  dialyzer  tube  of 
parchment  paper  for  twenty-four  hours  in  the  incubator.  At  the 
end  of  this  time  test  the  water  surrounding  the  dialyzer  for  re- 
ducing sugar  and  for  starch. 


IV.  MECHANISM  OF  SWALLOWING. 

1.  Inject  a  solution  of  0.03  gram  morphine  sulphate  under  the 
skin  of  a  medium-sized  rabbit.    Anaesthetize  lightly  with  ether. 
Tie  the  rabbit,  back  down,  upon  the  rabbit-board,  with  the  neck 
well  stretched  out.    Clip  and  shave  the  hair  in  the  neck  region  and 
also  over  the  epigastrium  and  zvphoid  appendix. 

2.  Make  a  median  incision  through  the  skin  and  fascia  of  the 
neck.    Separate  the  sterno-hyoid  muscles  and  expose  the  trachea. 
Carefully  separate  the  trachea  from  the  oesophagus,  which  lies  be- 
hind it,  avoiding  injury  to  the  nerves  and  vessels  running  beside 
and  between  the  two. 

3.  On  either  side  of  the  trachea  and  between  it  and  the  oesopha- 
gus, a  fine  nerve  filament  will  be  seen.    These  are  the  recurrent 
laryngeal  nerves.     Pass  a  loop  of  thread  around  each  recurrent 
laryngeal  nerve,  but  do  not  tie.    Isolate  the  vagus  nerve  of  each  side 
and  secure  with  untied  threads.    Follow  the  vagus  on  one  side  as 
far  as  the  level  of  the  lower  part  of  the  thyroid  cartilage.    At  this 
point  it  is  joined  by  the  superior  laryngeal  nerve.     Isolate  and 
pass  a  loop  of  thread  around  this  nerve. 

4.  Pass  a  heavy  ligature  around  the  trachea  as  low  in  the  neck 
as  practicable.    Cut  between  the  rings  of  the  trachea  just  above 
this  ligature.    Introduce  a  tracheal  cannula  and  tie  it  in  with  the 
ligature.    Excise  the  piece  of  trachea  between  the  cannula  and  the 
cricoid  cartilage.     This  brings  the  oesophagus  w^ell  into  view. 
Pass  two  threads  around  the  oesophagus,  to  be  tied  later. 

5.  Now  make  a  median  incision  through  the  abdominal  wall  in 
the  median  line  and  expose  the  stomach.    By  pulling  the  stomach 


LABORATORY  MANUAL  OF  PHYSIOLOGY. 

down  with  one  hand,  and  the  liver  and  lower  ribs  up  with  the  other, 
the  junction  of  the  oesophagus  and  stomach  may  be  seen. 

6.  Arrange  an  inductorium  for  weak  tetanizing  current.    Place 
the  electrodes  from  the  secondary  coil  under  the  superior  laryngeal 
nerve.    Arrange  a  metronome  or  chronograph  marking  seconds. 
This  is  to  assist  in  taking  the   time  of  the  swallowing  move- 
ments. 

The  work  of  the  experiment  may  be  divided  among  four  stu- 
dents as  follows : 

Let  one  student  manage  the  stimulation  of  the  superior  laryn- 
geal nerve  or  other  nerves  that  it  may  be  desired  to  stimulate  dur- 
ing the  course  of  the  experiment ;  let  another  make  the  time  ob- 
servations of  the  swallowing  movements;  let  a  third  manipulate 
the  stomach  for  observation  of  the  lower  end  of  the  oesophagus; 
and  let  a  fourth  make  careful  notes  of  the  observations. 

7.  Stimulate  the  superior  laryngeal  nerve  with  the  weak  tetan- 
izing current  until  the  rabbit  swallows.    Stimulation  of  this  afferent 
nerve  brings  about,  among  other  things,  a  reflex  swallow.    Note 
and  time  the  beginning  of  the  swallowing  movement.    Note  the 
passage  of  the  peristaltic  wave  along  the  cervical  portion  of  the 
oesophagus,  and  the  end  of  the  peristaltic  movement  at  the  stomach. 
How  much  time  has  elapsed  between  the  beginning  of  the  swallow 
and  the  ending  of  the  peristalsis  at  the  stomach  ?    Repeat  the  ob- 
servation a  number  of  times.    What  is  the  average  time  occupied 
by  the  passage  of  a  peristaltic  wave  over  the  length  of  the  oesopha- 
gus in  your  rabbit  ? 

8.  Determine  whether  the  mechanism  of  cesophageal  peristalsis 
is  a  nervous  reflex  one  or  due  to  muscular  conduction  of  the  con- 
traction wave  from  one  segment  of  the  oesophagus  to  another. 

Tie  two  ligatures  around  the  oesophagus,  in  the  cervical  region, 
and  cut  completely  through  the  gullet  between  the  ligatures.  Mus- 
cular continuity  is  thus  absolutely  severed. 

While  making  observations,  as  before,  produce  a  swallow  by 
stimulating  the  superior  laryngeal.  Does  the  peristaltic  wave  still 
pass  over  the  lower  segment  of  the  oesophagus  to  the  stomach  ?  If 


SECRETION— DIGESTION— ABSORPTION. 

so,  how  does  the  time  occupied  in  the  passage  of  the  wave  com- 
pare with  the  time  before  the  gullet  was  divided  ? 

If  the  peristaltic  wave  ceases  to  pass  below  the  cut,  what  con- 
clusion might  you  draw  concerning  the  method  of  conduction  of 
the  contraction  wave  ?  If  the  wave  continues  to  pass  after  the  sev- 
erance of  muscular  continuity,  what  conclusion  might  you  draw  ? 

9.  Secure  the  vagus  of  one  side  with  two  ligatures  and  cut  be- 
tween.   Again  induce  a  swallow  as  before.    Does  the  contraction 
wave  still  pass  over  the  oesophagus  ?    Explain. 

10.  Now  cut  the  other  vagus  also.    Both  vagus  nerves  are  now 
cut.     Induce  a  swallow.     Does  the  peristaltic  wave  continue  to 
pass  over  the  oesophagus  ? 

From  the  above  observations  what  conclusions  can  you  draw  con- 
cerning the  mechanism  of  cesophageal  peristalsis  and  the  function 
of  the  vagi  in  this  connection  ? 

11.  The  Vagus  as  a  Motor  Nerve  to  the  Stomach. — Enlarge 
the  abdominal  incision  so  as  to  expose  the  whole  of  the  stomach 
including  the  beginning  of  the  duodenum.    Place  the  peripheral 
ends  of  both  vagi  upon  electrodes  from  an  inductorium  arranged 
for  medium  strong  tetanizing  currents.    Stimulate  both  vagi  contin- 
uously and  note  the  strong  contraction  rings  which  pass  over  the 
stomach  from  the  fundus  toward  the  pylorus.    Note  the  opening  of 
the  pyloric  sphincter  and  the  expulsion  of  a  small  quantity  of  stom- 
ach content  into  the  duodenum.    Note  the  movements  of  the  duo- 
denum during  and  after  the  entrance  of  food  from  the  stomach. 
Keep  the  stomach  covered  with  a  pad  of  absorbent  cotton  moist- 
ened with  warm  physiological  saline,  between  observations. 

Over  what  part  of  the  stomach  wall  are  the  contraction  rings 
most  distinct  and  strongest?  Where  do  they  begin  and  in  what 
directions  do  they  pass  ? 

12.  Stimulate  both  inferior  laryngeal  nerves.    Note  the  effect 
upon  the  upper  segment  of  the  oesophagus.    What  is  the  nature  of 
the  musculature  of  the  first  part  of  the  oesophagus  ? 

13.  Free  the  small  piece  of  trachea  connected  with  the  cricoid 
cartilage  from  blood  and  note  the  position  of  the  vocal  bands. 

9  [I29l 


LABORATORY  MANUAL  OF  PHYSIOLOGY. 

Note  the  slight  opening  and  closing  of  the  glottis  with  inspiration 
and  expiration.  While  observing  the  movements  of  the  vocal 
bands,  stimulate  one  inferior  laryngeal  nerve.  What  is  the  effect 
on  the  vocal  bands?  Stimulate  both  nerves  at  the  same  time. 
What  is  the  effect  on  the  movements  of  the  vocal  bands  ? 

14.  Stimulate  the  superior  laryngeal  nerves  in  the  same  way  and 
note  the  effect,  if  any,  on  the  vocal  bands. 

15.  To  determine  the  time  occupied  for  the  passage  of  a  liquid 
from  the  mouth  into  the  stomach,  in  man,  proceed  as  follows: 

Arrange  a  drum  with  smoked  paper  for  medium  slow  revolution. 
Set  up  a  chronograph  to  mark  seconds  on  the  drum.  Place  a  short- 
circuiting  key  in  circuit  with  the  time  marker.  When  the  key  is 
closed,  the  lever  of  the  time  marker  will  write  a  straight  line.  When 
the  key  is  opened,  a  time  tracing,  in  seconds,  will  be  recorded. 
Take  a  fellow-student  into  a  quiet  room  and  listen  with  a  stetho- 
scope over  the  end  of  the  sternum.  Start  the  drum.  Let  the  sub- 
ject of  the  experiment  take  one  swallow  of  water.  You  will  hear 
two  sounds,  one  when  the  liquid  is  shot  into  the  oesophagus,  the 
other  when  the  liquid  enters  the  stomach.  When  the  first  sound 
is  heard,  open  the  short-circuiting  key.  When  the  second  sound  is 
heard,  close  the  key.  How  many  seconds  have  elapsed  between  the 
two  sounds  ? 

V.  GASTRIC  DIGESTION. 

1.  Tests  for  Proteids. — (a)  Coagulation  by  Heat. — Prepare 
solutions  of  the  following  proteids:  A,  egg  albumin,  dilute;  B, 
acid  albumin  in  acid  solution.  This  is  obtained  by  subjecting 
some  dilute  egg  albumin  to  the  action  of  o.2-per-cent  HC1  for  sev- 
eral hours  at  body  temperature.  Neutralizing  with  an  alkali  will 
precipitate  the  acid  albumin  from  its  solution. 

C,  myosin,  dissolved  in  a  lo-per-cent  NaCl  solution.  This  may 
be  prepared  by  mincing  lean  meat,  freeing  from  blood  by  repeated 
washings,  and  extracting  the  myosin  by  an  ammonium-chlorid 
solution.  The  salt  may  be  removed  by  dialysis,  leaving  the  myosin 
as  a  gelatinous  mass,  or  it  may  be  precipitated  by  diluting  the  solu- 


SECRETION— DIGESTION— ABSORPTION. 

tion  with  distilled  water.    This  precipitate  is  redissolved  in  the 
sodium-chlorid  solution  as  given  above. 

D,  proteose.    This  may  be  prepared  by  digesting  a  small  quan- 
tity of  fibrin  with  o.2-per-cent  HC1  and  a  little  commercial  pep- 
sin, at  38°  C.,  just  to  the  point  of  solution  of  the  fibrin  and  no  more. 
Neutralize  carefully  with  dilute  NaOH,  heat  to  boiling,  and  filter. 
Witte's  peptone  may  be  used,  since  it  consists  chiefly  of  albumoses. 

E,  peptone.    Savory  &  Moore's  preparation  is  used. 

Place  a  small  quantity  of  each  of  the  above  solutions  in  test- 
tubes  and  immerse  in  a  water-bath  heated  to  65°  C.  Gradually 
raise  the  temperature  of  the  bath  to  100°  C.,  noting  observations 
at  every  5°  rise  in  temperature. 

Are  all  of  these  solutions  coagulated  by  heat  ?  In  those  in  which 
coagulation  does  occur,  is  the  coagulating  point  the  same  ? 

(b)  Nitric-acid  Ring  Test.— Place  a  small  quantity  of  HNO3  in 
a  test-tube.    With  a  glass  tube  of  small  calibre  draw  up  some  of 
solution  A  into  the  tube,  and  with  the  finger  firmly  pressed  over  the 
other  end  introduce  the  end  containing  the  solution  into  the  acid. 
Remove  the  finger.    If  the  acid  level  is  slightly  higher  than  the 
level  of  the  liquid  in  the  tube,  some  of  the  acid  will  be  drawn  up 
into  the  glass  tube  containing  the  solution  to  be  tested.    Is  there 
any  ring  of  precipitation  formed  where  the  two  liquids  come  in 
contact  ?    Repeat  this  test  for  the  other  proteid  solutions  and  re- 
cord results. 

(c)  Xanthoproteic  Reaction. — Add   an   excess  of  concentrated 
nitric  acid  to  a  little  of  each  of  the  above  tested  solutions  and  heat 
to  boiling.    A  yellow  color  is  produced.    Neutralize  and  make  the 
solutions  alkaline  with  sodium  hydrate  or  ammonia.    The  color 
changes  to  an  orange  red. 

(d)  Biuret  Test. — Make  the  solutions  of  the  proteids  to  be  tested 
alkaline  with  sodium  hydrate.    Add  a  few  drops  of  a  dilute  cupric- 
sulphate  solution.    Be  careful  not  to  add  an  excess  of  the  copper 
solution,  since  this  may  give  a  test  in  the  absence  of  proteid.    A 
blue-purple  or  violet  color  results. 

(e)  Millon's  Test. — Test  each  of  the  solutions  with  Millon's 


LABORATORY  MANUAL  OF  PHYSIOLOGY. 

reagent,  which  is  already  made  up.  This  is  a  solution  of  mercu- 
rous  nitrate  together  with  some  free  nitrous  acid.  When  mixed 
with  proteid  a  yellow  precipitate  is  formed  which  becomes  red  on 
heating. 

2.  To  Differentiate  Albumins,  Proteoses,  and  Peptones.— 
(a)  What  reactions  have  they  in  common  ?   Are  the  albumins  co- 
agulable  by  heat?    Are  the  proteoses  coagulated  D>  neat?    Are 
the  peptones  coagulated  by  heat  ? 

(b)  Do  the  proteoses  give  a  precipitate  with  nitric  acid?    Do 
the  peptones? 

(c)  To  a  proteose  solution  add  some  potassium  ferrocyanid  acid- 
ified with  acetic  acid.    Is  there  any  precipitate?    Repeat  with  a 
pure  peptone  solution.    Is  there  any  precipitate  ? 

(d)  To  a  solution  of  proteoses  add  sodium  chlorid  to  satura- 
tion.   Is  there  any  precipitate  ?    Repeat  with  pure  peptone  solution. 

(e)  To  a  peptone  solution  add  alcohol.    Is  a  precipitate  formed  ? 
Repeat  using  a  saturated  solution  of  tannic  acid  instead  of  the 
alcohol.    Result  ? 

3.  Artificial  Gastric  Juice. — Scrape  off  the  mucous  membrane 
of  the  fresh  stomach  of  a  pig.    Grind  this  thoroughly  with  clean 
sand  in  a  mortar.    Add  ten  times  the  volume  of  a  o.2-per-cent  so- 
lution of  hydrochloric  acid  and  place  in  the  incubator  at  blood 
temperature  for  twenty-four  hours. 

Grind  up  another  portion  of  mucous  membrane  of  the  pig's 
stomach  with  glycerin.  Let  this  stand  for  several  days  before 
using. 

(a)  To  a  little  fibrin  in  a  test-tube  add  some  o.2-per-cent  HC1. 

(b)  To  another  portion  of  fibrin  add  some  of  the  glycerin  ex- 
tract of  the  pig's  stomach. 

(c)  To  another  portion  of  fibrin  add  some   of  the  acidulated 
aqueous  extract  of  pig's  stomach. 

(d)  To  another  portion  add  some   of  the  acidulated  extract 
which  has  been  neutralized  and  made  slightly  alkaline  with  so- 
dium carbonate. 

(e)  To  another  portion  add  some  glycerin  extract  plus  enough 


SECRETION— DIGESTION— ABSORPTION. 

o.2-per-cent  HC1  to  make  the  acidity  of  the  mixture  equal  to 
about  o.i  per  cent  HC1. 

(/)  To  another  portion  add  glycerin  extract  plus  o.4-per-cent 
acetic  acid. 

(g)  To  another  portion  add  glycerin  extract  plus  o.4-per-cent 
lactic  acid. 

Make  the  fibrin  portion  in  each  tube  as  near  the  same  quantity 
as  possible  and  use  the  same  amount  of  the  extract  each  time. 
Place  all  the  tubes,  properly  labelled,  in  a  water-bath  kept  at  a 
temperature  of  38°  C.  Examine  the  specimens  every  three  to  five 
minutes,  and  note  the  extent  of  the  solution  of  the  fibrin  in  each 
tube.  Continue  the  observations  for  a  half-hour.  Solution  of  the 
fibrin  is  not  an  index  of  complete  digestion,  but  is  a  sufficient  index 
for  rough  comparison  of  the  digestive  activity  of  the  mixtures  in 
the  various  tubes. 

Note  results  and  record  observations.  The  glycerin  extract 
contains  pepsin,  but  no  acid.  What  is  the  digestive  activity  of  pep- 
sin in  the  absence  of  acid  ?  What  is  the  digestivity  of  HC1  alone, 
in  the  absence  of  pepsin  ?  Is  there  any  digestion  with  the  alkaline 
mixture  of  pepsin?  Does  digestion  occur  when  other  acids  are 
substituted  for  HC1  ? 

(h)  Boil  some  of  the  acidulated  glycerin  extract.  Add  this  to 
fibrin  and  note  results.  Does  heat  destroy  the  enzyme  ? 

4.  Filter  the  contents  of  one  or  more  of  the  tubes  in  which  the 
fibrin  has  been  dissolved. 

(a)  To  a  portion  of  this  filtrate  add  dilute  NaOH,  carefully, 
until  the  acid  is  neutralized.    Is  there  any  precipitate  ?    If  so,  the 
presence  of  what  proteid  is  indicated  ?    Filter. 

(b)  To  a  part  of  this  filtrate  add  an  excess  of  the  alkali  and 
then  a  drop  or  two  of  very  dilute  CuSO4  solution.    How  is  the  pres- 
ence of  proteoses  and  peptones  indicated  ?    What  is  the  necessity 
for  care  in  adding  the  copper-salt  solution? 

(c)  Heat  another  part  of  the  filtrate  from  (a)  to  70°  C.    Is  there 
any  coagulation  ? 

(d)  Take  another  portion  of  the  filtrate  from  (a)  and  saturate 

[133] 


LABORATORY  MANUAL  OF  PHYSIOLOGY. 

the  solution  with  ammonium  sulphate.  How  is  the  presence  of 
proteoses  indicated  ?  How  are  they  differentiated  from  peptones  ? 

(e)  Filter  portion  (d)  and  test  the  nitrate  for  the  biuret  reaction. 
If  present,  what  does  it  indicate  ? 

(/)  Take  a  portion  of  the  fibrin  which  has  been  acted  upon  by 
the  acidulated  glycerin  extract,  neutralize  the  mixture  with  sodium 
carbonate,  and  place  in  a  dialyzer  over  night.  The  next  day  test 
the  dialyzant  for  albumin,  proteoses,  and  peptones. 

Test  the  fluid  remaining  in  the  dialyzer  in  the  same  way.  Re- 
sults ?  Conclusions  ? 

5.  Place  some  fresh  milk  in  a  test-tube,  in  a  water-bath  at  38°  C., 
and  add  a  few  drops  of  rennet  extract.  What  is  the  action  of  the 
rennin  ?  Remove  the  fluid  part  of  the  milk  so  far  as  possible,  and 
add  some  artificial  gastric  juice.  Is  the  casein  dissolved  ? 

VI.  INTESTINAL  DIGESTION. 

1.  Emulsification. — (a)  Shake  up  5  c.c.  of  olive  oil  in  a  test- 
tube  with  an  equal  quantity  of  water.    The  mixture  will  become 
milky  white  because  of  the  distribution  of  oil  globules  through  it. 
Do  these  oil  globules  remain  in  suspension  ? 

(b)  Repeat  the  procedure  in  (a),  adding  to  the  mixture,  before 
shaking,  5  c.c.  of  strained  egg  albumin.    Do  the  oil  globules  remain 
in  suspension  ?    Set  this  to  one  side  and  observe  again,  some  hours 
later.    Has  any  of  the  oil  begun  to  separate  out  ? 

(c)  Repeat  (6),  substituting  gum  acacia  for  the  egg  albumin. 
Set  aside  and  observe  later  for  the  separation  of  oil  from   the 
emulsion. 

2.  Saponification. — To  5  c.c.  of  olive  oil  or  cottonseed  oil  add 
twice  the  volume  of  a  20-per-cent  solution  of  NaOH  or  KOH. 
Shake  well.    Continue  to  agitate  the  mixture  at  frequent  intervals 
for  fifteen  to  twenty  minutes.    Now  add  an  excess  of  water.    Has 
the  oil  been  dissolved  ?    What  chemical  reaction  has  taken  place 
between  the  oil  and  the  alkali  ? 

3.  Saponification   as   an  Aid   in  Emulsification. — (a)  Mix 
a  small  quantity  of  oil  which  has  become  slightly  rancid,  with  a 


SECRETION— DIGESTION— ABSORPTION. 

strong  solution  of  sodium  carbonate.  Shake  the  mixture  vigor- 
ously. Set  the  emulsion  thus  formed  aside  and  compare  it  later 
with  the  emulsions  made  with  egg  albumin  and  gum  acacia. 

(b)  Repeat  (a),  but  do  not  shake  the  mixture.  Is  an  emulsion 
produced  ?  Place  some  of  this  mixture  under  the  microscope  and 
observe  the  disintegration  of  the  oil-drops  into  small  globules. 
How  does  this  compare  with  the  emulsification  of  fats  in  the  intes- 
tine? 

4.  A  pancreatic  extract  may  be  made  in  the  following  way. 
Take  a  pig's  pancreas  or  a  dog's  pancreas  which  has  lain  for 
twenty-four  hours  at  the  room  temperature.  Cut  into  small 
pieces  or  run  through  a  meat  grinder.  Crush  in  a  mortar  with 
twice  its  volume  of  glycerin.  Place  the  mixture  in  a  bottle  and 
allow  to  stand  for  several  days  before  using. 

For  use,  strain  the  mixture  through  a  fine  cloth  and  dilute  as 
needed  with  four  or  five  times  its  volume  of  water  containing  so- 
dium carbonate  to  make  the  mixture  distinctly  alkaline.  Prepare 
a  series  of  test-tubes  for  digestion  experiments  as  follows : 

A .  Place  a  small  quantity  of  fibrin  in  a  test-tube,  adding  the 
alkaline  diluted  glycerin  extract  of  pancreas  until  the  tube  is  half 
full. 

B.  To  another  tube  containing  fibrin  add  the  glycerin  extract 
diluted  with  water,  alone,  and  without  the  addition  of  the  alkali. 

C.  To  another  portion  of  fibrin  add  glycerin  extract  of  pan- 
creas and  dilute  with  o.i-per-cent  HC1. 

D.  To  another   portion   of  fibrin   add  some   glycerin  extract 
which  has  been  boiled,  and  dilute  with  o.i-per-cent  sodium  car- 
bonate. 

E.  To  another  fibrin  portion  add  o.i-per-cent  sodium  carbon- 
ate, alone. 

Prepare  a  second  series  of  tubes  in  the  same  way,  substituting 
commercial  pancreatin  for  the  glycerin  extract. 

Place  all  these  tubes  in  a  water-bath  at  a  temperature  of  38°  C. 
From  time  to  time  make  an  observation  of  the  changes  which  may 
be  going  on  in  the  various  tubes.  After  a  half -hour,  in  which  tubes 


LABORATORY  MANUAL  OF  PHYSIOLOGY. 

has  solution  of  the  fibrin  taken  place  ?  At  the  end  of  an  hour,  what 
is  the  condition  of  the  fibrin  in  the  various  tubes  ? 

In  those  tubes  in  which  digestion  of  the  fibrin  has  taken  place, 
what  difference  can  be  made  out,  by  direct  observation,  between 
the  action  of  pancreatic  and  gastric  juice  in  their  attack  upon  pro- 
teids? 

Filter  the  contents  of  those  tubes  which  have  shown  digestive 
change  and  test  for  albumins,  proteoses,  and  peptones. 

Place  some  of  the  filtrate  in  a  dialyzer,  and  the  following  day  test 
the  fluid  surrounding  the  dialyzer  for  proteoses  and  peptones. 

5.  Amylolytic  Action  of   Pancreatic  Juice. — (a)  Make  up 
some  starch  paste.    Test  it  with  Fehling's  solution  to  make  sure  of 
the  absence  of  a  reducing  sugar.    Mix  some  of  this  paste  with  dilute 
pancreatic  extract  of  neutral  reaction.    Label  this  tube  A. 

In  a  second  tube  of  starch  paste,  B,  place  pancreas  extract  of  al- 
kaline reaction. 

To  a  third  tube  of  starch  paste  add  pancreas  extract  made  acid 
with  o.i-per-cent  HC1.  Label  this  tube  C. 

To  a  fourth  portion  of  starch  paste  add  o.i-per-cent  HC1 
alone.  Label  D. 

To  a  fifth  portion  add  strong  hydrochloric  acid.    Label  E. 

To  a  sixth  portion  add  o.i-per-cent  sodium-carbonate  solution. 
Label  F. 

Place  all  these  tubes  in  the  water-bath  at  38°  C.  In  ten  or  fifteen 
minutes  test  for  reducing  sugar  in  all  the  tubes,  first  carefully  neu- 
tralizing those  of  an  acid  reaction.  Record  results.  In  which 
tubes  has  starch  digestion  taken  place  ?  What  is  the  starch-digest- 
ing enzyme  of  the  pancreatic  secretion  ?  What  is  the  optimum  re- 
action of  the  digesting  medium  ? 

(b)  Mix  some  starch  paste  with  pancreatic  extract  which  has 
been  previously  boiled.  Place  in  the  warm  water-bath  and  note 
results.  W7hat  is  the  effect  of  high  temperature  on  the  enzyme  ?  Is 
this  effect  common  to  all  enzymes  ? 

6.  Lipolytic  Action  of  Pancreatic  Juice. — Mix  a  small  quan- 
tity of  fresh  butter  in  a  test-tube  with  glycerin  extract  of  pancreas 


SECRETION— DIGESTION— ABSORPTION. 

and  o.i-per-cent  sodium  carbonate.  Place  the  mixture  in  the  warm 
water-bath.  What  change  occurs  in  the  butter  ?  After  a  time  can 
you  detect  the  odor  of  butyric  acid  ?  If  so,  what  is  its  significance  ? 

7.  Action  of  Bile. — You  will  be  supplied  with  ox-bile.    What 
is  its  reaction  ? 

(a)  Test  for  bile  pigments  as  follows  (Gmelin's  reaction) :   To 
a  little  bile  on  white  porcelain  add  a  few  drops  of  fuming  yellow 
nitric  acid.    Note  the  changes  in  color  from  green  to  blue,  yellow, 
and  brown-yellow.    The  test  may  also  be  done  by  placing  a  drop 
of  bile  on  white  filter  paper  and  bringing  a  drop  of  the  acid  in 
contact  with  it.    Color  rings  will  be  formed  at  the  junction  of  the 
bile  and  the  acid. 

(b)  Pettenkojer's  Test  for  Bile  Acids. — Mix  some  ox-bile  in  a  test- 
tube  with  a  small  amount  of  strong  sulphuric  acid.    Test  the  tem- 
perature of  the  mixture  with  a  thermometer,  adding  the  acid 
slowly.    The  temperature  should  not  be  higher  than  70°  C.  or  lower 
than   50°  C.     Now  add  a  ic-per-cent  solution  of   cane  sugar, 
slowly,  drop  by  drop,  stirring  with  a  glass  rod.    A  red  coloration 
indicates  the  presence  of  bile  acids.    This  reaction  is  masked  by 
using  an  excess  of  sugar  or  too  high  a  temperature,  since  the  sugar 
is  decomposed  and  colors  the  mixture  a  dark  brown. 

(c)  Mix  some  fresh  butter  in  a  test-tube  with  a  few  cubic  centi- 
metres of  ox-bile.    Mix  a  portion  of  butter  in  another  tube  with 
bile  and  pancreatic  extract.    Place  in  the  warm  water-bath  and 
note  results. 

8.  Absorption  of  Fat. — Starve  a  cat  for  twenty-four  hours  and 
kill.    Open  the  abdomen  and  note  the  condition  of  the  mesenteric 
lymphatics,  the  lacteals.    Open  a  loop  of  small  intestine.    Scrape  off 
some  of  the  mucous  membrane.    Tease  some  of  the  scrapings,  on  a 
slide,  with  glycerin,  after  previous  immersion  in  osmic  acid  J  per 
cent  for  twenty-four  hours.    Remove  the  excess  of  glycerin  with 
filter  paper,  and,  covering  the  preparation  with  a  cover  slip,  ex- 
amine under  the  microscope.    If  fat-droplets  are  present  in  the 
epithelium,  they  will  be  stained  black  or  dark  brown  by  the  osmic 
acid.    Note  results. 


LABORATORY  MANUAL  OF  PHYSIOLOGY. 

Feed  another  cat,  after  a  starvation  period  of  twelve  hours,  with 
fatty  meat  or  milk  and  cream.  Kill  in  ten  hours  after  the  meal. 
Prepare  the  intestinal  mucous  membrane,  as  before,  with  osmic 
acid.  Also  note  the  appearance  of  the  lacteals.  Can  you  demon- 
strate the  presence  of  fat  in  the  intestinal  epithelium  ? 

VII.  MECHANISM  OF  PANCREATIC  SECRETION. 
(DEMONSTRATION.) 

i.  Narcotize  a  medium-sized  dog  with  morphine  sulphate  and 
anaesthetize  with  ether.  Expose  and  introduce  a  cannula  into  the 
trachea.  Expose,  isolate,  and  secure  both  vagus  nerves  with  un- 
tied ligatures.  Connect  the  tracheal  cannula  with  the  artificial  re- 
spiratory apparatus  and  anaesthetic  flask.  Through  a  median  ab- 
dominal incision  expose  the  duodenum  and  head  of  the  pancreas. 
Slit  the  duodenum  longitudinally  for  an  inch  or  more  in  the  neigh- 
borhood of  the  head  of  the  pancreas.  Place  a  cannula  in  the  larger 
pancreatic  duct  through  its  duodenal  opening,  on  a  level  with  the 
lower  border  of  the  pancreas.  Connect  this  with  a  long  glass  tube, 
bent  so  as  to  pass  over  the  edge  of  the  abdominal  wound.  Fill  this 
tube  with  physiological  salt  solution. 

To  the  rubber  membrane  of  a  transmitting  tambour  cement  a 
light  aluminum  shovel-shaped  lever.  Arrange  the  transmitting 
tambour  in  connection  with  a  recording  and  a  slow  drum.  Bring 
the  end  of  the  pancreas-cannula  tube  over  the  shovel  lever  of  the 
tambour.  Each  drop  from  the  end  of  the  cannula  will  then  depress 
the  lever  of  the  transmitting  tambour,  and  this  will  be  transmitted 
to  the  recording  tambour  and  a  record  will  be  written  on  the  paper 
of  the  revolving  drum. 

During  the  continuation  of  the  experiment  the  exposed  abdom- 
inal viscera  should  be  protected  by  cotton  pads  soaked  in  warm 
o.8-per-cent  NaCl  solution. 

After  completion  of  the  operative  procedures,  note  the  rate  of 
flow  of  pancreatic  juice  for  a  period  of  ten  or  fifteen  minutes.  Now 
inject  into  the  duodenum  or  jejunum  30  c.c.  of  a  o.4-per-cent  HC1 


SECRETION— DIGESTION— ABSORPTION. 

solution.  Note  the  rate  of  pancreatic  secretion  for  another  period 
of  fifteen  minutes. 

Allow  ten  minutes'  rest.  Then  divide  both  vagus  nerves  and 
stimulate  with  a  weak  tetanizing  current.  Note  any  change  in  the 
rate  of  pancreatic  flow. 

Repeat  the  injection  of  the  acid.  Is  the  same  effect  produced 
that  occurred  after  the  first  injection  ? 

2.  Action  of  Secretin  (Bayliss  and  Starling). — That  the  chief 
stimulus  to  the  elimination  of  pancreatic  secretion  is  a  chemical 
one  was  demonstrated  by  Bayliss  and  Starling.  They  have  found 
that  there  is  a  substance  present,  in  the  mucous  membrane  of 
the  upper  part  of  the  small  intestine  chiefly,  which  they  have 
termed  prosecretin.  This,  in  the  presence  of  hydrochloric  acid,  or 
other  acids  to  a  less  extent,  is  changed  to  another  substance,  se- 
cretin,  which  is  absorbed,  passes  to  the  pancreas,  and  there  acts  as 
a  stimulus  to  the  elimination  of  pancreatic  juice. 

An  active  secretin  extract  is  prepared  as  follows:  Cut  out  a  dog's 
duodenum  and  jejunum.  Slit  open  the  bowel.  Wash  thoroughly 
with  water.  Scrape  off  the  mucous  membrane.  Grind  this  with 
sand  and  a  little  o.4-per-cent  HC1  in  a  mortar.  Add  three  times  its 
volume  of  o.4-per-cent  HC1  and  allow  to  stand  for  fifteen  to  twenty 
minutes.  Bring  to  a  boil  in  a  porcelain  capsule,  and  while  boiling 
neutralize  and  render  slightly  alkaline  with  strong  NaOH.  Acidify 
slightly  with  acetic  acid,  strain  through  muslin,  and  filter. 

Isolate  and  introduce  a  cannula  into  the  central  end  of  the  fem- 
oral vein  of  the  dog  of  experiment  i.  While  a  record  of  flow  of 
the  pancreatic  juice  is  being  taken,  introduce  into  the  vein  5  c.c. 
of  the  secretin  extract  prepared  as  described  above.  Note  the  re- 
sult on  the  rate  of  elimination  of  the  pancreatic  secretion. 

Repeat  the  injection  several  times,  allowing  a  period  of  rest  be- 
tween injections. 


CHAPTER  VII. 
INTERNAL   SECRETIONS. 

I.  LIVER,  GLYCOGEN. 

i.  SELECT  a  well-nourished  rabbit.  Kill  quickly  by  a  sudden 
blow  upon  the  back  of  the  neck  in  the  region  of  the  medulla.  As 
speedily  as  possible,  open  the  abdomen  and  remove  a  portion  of  the 
liver.  Cut  this  into  small  pieces.  Place  some  of  these  pieces  in  boil- 
ing water;  and  place  one  piece  immediately  on  the  holder  of  a 
freezing  microtome  and  freeze  the  piece. 

(a)  Make  a  number  of  sections  with  the  freezing  microtome,  and 
as  soon  as  cut  place  in  Lugol's  solution  containing  iodine  and  po- 
tassium iodide.    Allow  the  sections  to  remain  in  the  staining  solu- 
tion for  two  or  three  minutes.    Wash  in  water  to  remove  excess  of 
the  iodine  solution.    Mount  in  glycerin  and  examine  under  the 
microscope. 

The  glycogen  present  in  the  liver  cells  will  be  stained  a  mahog- 
any red  by  the  iodine.  Compare  this  reaction  with  that  given  by 
dextrin  with  iodine. 

(b)  Allow  some  of  the  rabbit's  liver  to  remain  in  the  incubator 
at  body  temperature  for  an  hour  or  longer.    Cut  frozen  sections  of 
a  piece  of  this  and  treat  with  iodine  as  before.    Is  there  any  gly- 
cogen reaction  ? 

(c)  Grind  some  of  the  incubated  liver  with  sand  in  a  mortar. 
Add  two  or  three  volumes  of  water.    Allow  to  stand  for  several 
minutes.    Strain  through  muslin  and  filter  through  paper,  and  test 
for  reducing  sugar  with  Fehling's  solution. 

(d)  To  prepare  glycogen,  take  the  pieces  of  liver  which  have 
been  immersed  in  the  boiling  water,  grind  them  in  a  mortar  with 

[140] 


INTERNAL  SECRETIONS. 

fine  sand,  return  to  the  capsule,  and  boil  again  for  a  short  time  to 
make  certain  that  the  diastatic  power  of  the  liver  has  been  de- 
stroyed. Add  ten  volumes  of  water  slightly  acidulated  with  acetic 
acid.  Strain  through  muslin.  To  remove  the  proteids,  concen- 
trate the  liquid  to  a  third  its  volume  and  add  alternate  drops  of 
HCl  and  potassium  mercuric  iodid  until  precipitation  ceases. 
Filter  off  a  little  of  the  liquid  and  test  the  filtrate  for  proteids.  If 
there  are  none  present,  strain  all  of  the  liquid  through  muslin  and 
filter  through  paper.  To  the  filtrate  add  two  volumes  of  alcohol, 
stirring  well.  Allow  the  precipitate,  glycogen,  to  settle,  decant  off 
the  supernatant  liquid,  filter  the  residue,  and  wash  with  dilute  al- 
cohol. Transfer  the  residue  to  a  beaker,  cover  with  absolute  alco- 
hol, and  set  aside  for  an  hour.  Remove  the  alcohol  and  dry  the 
residue  between  folds  of  filter  paper. 

To  some  of  the  glycogen  thus  prepared  add  25  c.c.  of  water  and 
warm  gently.  A  solution  is  formed.  Compare  the  appearance  of 
this  solution  with  that  of  soluble  starch. 

Test  a  little  of  the  glycogen  solution  with  iodine  and  KI.  Note 
the  color  reaction.  Does  it  disappear  upon  heating  ?  If  so,  does 
it  reappear  upon  cooling  ? 

Test  some  of  the  solution  of  glycogen  with  Fehling's  reagent. 
Is  there  any  reduction  of  the  copper  salt  brought  about  ? 

To  a  little  of  the  glycogen  solution  add  some  saliva.  After  a  few 
minutes  test  with  Fehling's  reagent  for  reducing  sugar. 

II.  PANCREATIC  DIABETES. 

Effect  of  Removal  of  the  Pancreas  on  Carbohydrate  Metabolism. — 
Collect  the  urine  of  a  medium-sized  dog  and  test  for  reducing  sugar 
with  Fehling's  solution.  In  all  probability  none  will  be  found. 
Starve  the  dog  for  twelve  hours.  Inject  subcutanrously  0.12  gram 
of  morphine  sulphate.  Place  dog,  back  down,  on  the  operating- 
board  and  continue  the  anaesthesia  with  ether.  Prepare  sterile 
medium  and  heavy  silk  suture  material.  Sterilize  a  number  of 
absorbent  cotton  pads  in  physiological  salt  solution.  Sterilize 
instruments  by  steam  or  by  boiling. 


LABORATORY  MANUAL  OF  PHYSIOLOGY. 

Clip  and  shave  the  hair  over  the  *  midabdominal  region  and 
wash  the  skin  with  corrosive  sublimate,  i  to  1000,  followed  by  al- 
cohol. Clean  the  hands  by  thorough  scrubbing  and  immersion  in 
the  corrosive-sublimate  solution. 

Open  the  abdomen  by  an  incision  in  the  median  line.  Locate 
the  head  of  the  pancreas  at  the  duodenum  and  the  tail  at  the  spleen. 
Carefully  separate  the  pancreas  from  the  omentum  and  mesentery 
and  from  the  duodenum,  tying  the  pancreatic  ducts  and  all  ar- 
teries and  veins  with  double  ligatures  and  cutting  between. 
Having  controlled  hemorrhage  in  this  way,  remove  the  pancreas 
in  tola. 

Bring  the  peritoneum  and  abdominal  muscles  together  with  one 
row  of  interrupted  sutures  and  close  the  skin  wound  with  a  second 
row  of  sutures.  Dry  the  wound  with  alcohol  and  ether  and  paint 
with  collodion. 

After  the  animal  has  recovered  from  the  anaesthetic  and  the  im- 
mediate shock  of  the  operation,  collect  the  urine  every  six  hours  and 
make  quantitative  estimations  of  the  sugar  by  Fehling's  method. 
Also,  carefully  note  the  condition  of  the  dog,  as  to  loss  of  weight, 
temperature,  appetite,  weakness,  convulsions,  etc.  The  diet  may 
be  the  same  as  that  given  previous  to  the  operation.  Make  an  es- 
timate of  the  carbohydrates,  proteids,  and  fat  of  the  diet. 

If  the  dog  survives  more  than  forty-eight  hours  after  the  oper- 
ation, the  diet  may  be  changed  to  pure  proteid  and  a  known 
amount  of  raw  pancreas  given  with  the  food. 

Immediately  after  the  death  of  the  animal,  the  liver  should  be 
removed  and  tested  for  glycogen  as  described  for  experiment  I. 

III.  THYROID. 

1.  Ablation  of  the  Thyroid  in  Dogs.— This  is  best  done  in 
two  operations.  At  the  first  operation  one  lobe  of  the  thyroid  is 
removed,  and  at  the  second  the  other  lobe. 

Select  a  young  dog.  Record  weight.  Place  under  morphine 
narcosis.  Shave  and  scrub  skin  of  the  neck  and  wash  with  a  bi- 
chloride solution,  i  to  icoo.  The  operation  should,  of  course,  be 


INTERNAL  SECRETIONS. 

done  with  strict  asepsis.  The  instruments  should  be  boiled,  liga- 
tures sterilized,  and  hands  cleansed  and  soaked  in  the  bichloride 
solution. 

Expose  the  trachea  through  a  median  cervical  incision,  carrying 
the  incision  as  far  as  the  thyroid  cartilage.  Pull  aside  and  sepa- 
rate, by  blunt  dissection,  the  longitudinal  neck  muscles  from  the 
thyroid  lobe  of  one  side.  This  is  seen  as  an  oval,  reddish  mass. 
With  blunt  hooks  and  scalpel  handle  separate  it  from  its  attach- 
ments. Tie  all  blood-vessels  with  two  ligatures  and  cut  between. 
The  thyroid  branch  of  the  carotid  artery  should  not  be  tied  too 
near  its  origin,  since  there  is  danger  of  the  ligature  slipping  later, 
and  secondary  hemorrhage.  If  an  isthmus  is  present  connecting 
the  two  lateral  lobes,  this  also  should  be  removed. 

The  wound  is  now  cleansed  with  sterile  o.8-per-cent  NaCl  solu- 
tion, dried,  and  closed  by  two  rows  of  interrupted  silk  sutures,  one 
to  draw  the  muscles  together  over  the  trachea,  the  other  to  approx- 
imate the  skin.  Cover  the  wound  with  a  thin  layer  of  sterile  ab- 
sorbent cotton  and  paint  with  collodion. 

Keep  the  animal  under  careful  observation  for  ten  days  or  two 
weeks,  recording  the  weight  daily.  At  the  end  of  this  time,  repeat 
the  operative  procedure  and  remove  the  remaining  thyroid  lobe. 
Keep  under  careful  observation,  recording  the  weight  daily,  taking 
the  temperature  per  rectum,  and  counting  the  red  and  white  cor- 
puscles of  the  blood.  Note  all  symptoms  and  keep  a  careful  record 
until  death  occurs.  If  the  animal  does  not  die  or  show  the  charac- 
teristic symptoms  of  thyroid  removal,  all  the  thyroid  has  not  been 
removed  or  there  are  accessory  thyroids  present.  This  may  be  de- 
termined at  autopsy  later. 

At  autopsy  the  condition  of  all  the  organs  should  be  determined 
and  the  field  of  the  operation  examined  to  see  if  the  thyroid  re- 
moval was  complete  and  if  accessory  bodies  are  present.  These 
are  sometimes  found  in  the  neck  region  near  the  thyroid  lobes 
proper,  or  in  the  mediastinum.  If  found,  they  should  be  hardened, 
embedded,  sections  cut,  stained,  and  examined  for  thyroid  struct- 
ure 


LABORATORY  MANUAL  OF  PHYSIOLOGY. 

2.  Thyroid  Feeding  after  Thyroid  Removal. — Remove  the 
thyroid  of  another  dog  in  the  same  way  as  in  experiment  i ,  and 
at  about  the  same  time,  so  that  the  symptoms  of  the  two  animals 
may  be  compared.  The  second  dog  should  be  fed,  after  the  com- 
plete removal  of  the  gland,  with  fresh  sheep's  thyroids,  or  thyroid 
extracts  may  be  mixed  with  the  food. 

What  are  the  symptoms  of  thyroid  removal  in  the  dog  ?  How  d< 
they  compare  with  those  stated  to  occur  in  similar  conditions  in 
man  ?  How  do  they  compare  with  the  symptoms  of  thyroid  dis- 
ease in  man?  Has  thyroid  feeding  any  effect  in  alleviating  the 
symptoms  following  thyroid  removal  in  the  dog  ? 

If  the  dog  fed  with  thyroids  survives,  it  should  be  killed  later 
and  careful  search  made  for  accessory  bodies. 

IV.  SUPRARENAL  GLANDS. 

1.  Ablation  of  the  Suprarenal  in  the  Rabbit. — Demonstra- 
tion.— Select  a  large,  well-nourished  rabbit.  The  Belgian  hare 
serves  well  for  the  purpose.  Starve  for  twenty-four  hours.  The 
operation  is  done  in  two  stages,  one  suprarenal  being  removed  at 
the  first  operation,  and  the  other  two  weeks  later.  The  left 
suprarenal  is  removed  most  readily  by  the  abdominal  route.  The 
right  suprarenal  is  reached  best  by  the  dorsal  route  without  enter- 
ing the  peritoneal  cavity. 

Inject  under  the  skin  9  cgm.  of  morphine  sulphate.  Strap  the 
rabbit,  back  down,  upon  the  rabbit-board.  Clip  and  shave  the  hair 
from  the  midabdominal  region.  Wash  with  bichlorid.  Sterilize 
instruments  by  boiling.  Sterilize  absorbent  cotton  pads  in  o.8-per- 
cent  NaCl  solution. 

Beginning  just  below  the  xyphoid  appendix  of  the  sternum, 
make  an  incision  in  the  midabdominal  line,  through  the  skin, 
fascia,  and  peritoneum.  The  length  of  the  incision  should  be 
about  three  inches.  Cover  the  intestines  and  hold  them  back  with 
absorbent  cotton  pads  wrung  out  in  hot  salt  solution.  Let  an  as- 
sistant hold  back  the  edges  of  the  abdominal  wound  with  retrac- 
tors. Locate  the  left  kidney.  Follow  the  renal  vein  to  its  junction 


INTERNAL  SECRETIONS. 

with  the  inferior  vena  cava.  A  little  above  the  angle  formed  by 
these  two  veins  and  closely  hugging  the  inferior  vena  cava,  the  left 
suprarenal  capsule  will  be  seen. 

This  is  a  yellowish-white  oval  body,  lying  behind  the  perito- 
neum. Its  blood  supply  is  variable.  There  are  generally  one  large 
artery  and  vein  entering  the  hilum  of  the  gland,  and  these  must  be 
tied  before  the  gland  is  excised. 

With  small  blunt  hooks  and  a  blunt-pointed  seeker  tear  through 
the  overlying  peritoneum.  Separate  the  gland  from  its  surround- 
ings slowly  and  carefully,  being  especially  careful  to  avoid  injury 
to  the  inferior  vena  cava. 

Remove  all  cotton  pads  from  the  abdominal  cavity.  Bring  the 
muscles  and  peritoneum  together  with  one  row  of  silk  sutures,  and 
the  skin  with  another  row  of  sutures.  Cover  the  wound  with  some 
antiseptic  powder  and  apply  a  gauze  bandage.  Place  the  rabbit  in 
its  cage  and  observe  it  carefully  twice  a  day  for  the  first  two  days 
and  then  make  daily  observations.  At  the  end  of  two  weeks  the 
remaining  gland  may  be  removed.  In  the  mean  time  make  an  ex- 
tract of  the  gland  as  described  below. 

2.  Weigh  the  suprarenal  which  you  have  removed.    Grind  it  in  a 
mortar  with  fine  clean  sand  and  a  little  o.y-per-cent  NaCl  solu- 
tion.    Add  of  the  salt   solution  enough  to  equal  ten  times  the 
weight  of  the  gland.    Transfer  to  a  tightly  stoppered  bottle  and 
place  in  the  incubator  at  30°  C.  for  fifty-six  hours.    Strain  through 
muslin  and  filter  through  paper.    One  part  of  this  extract  will  be 
equivalent  to  y1^  of  the  fresh  gland. 

3.  (a)  Prepare  another  rabbit  for  a  blood-pressure  experiment. 
Isolate  both  vagi  and  secure  them  with  untied  ligatures.    Prepare 
one  jugular  vein  for  injection.     Take  a  normal  blood-pressure 
tracing.    While  the  tracing  is  being  recorded,  inject  into  the  jug- 
ular  enough  of  the  suprarenal  extract  to  be  equivalent  to  -f^ 
of  the  fresh  gland.    Note  the  effect  on  the  heart-beat  and  on 
blood  pressure.    What  is  the  duration  of  the  effect  of  the  in- 
jection ? 

(b)  Repeat  the  injection,  and  immediately  after  cut  both  vagus 

10  [  US  ] 


LABORATORY  MANUAL  OF  PHYSIOLOGY. 

nerves.  Compare  the  effect  of  the  second  injection  upon  heart- 
beat and  blood  pressure  with  the  result  of  the  first  injection,  when 
the  vagus  nerves  were  intact. 

(c)  Repeat,  using  in  place  of  the  extract  of  the  rabbit's 
suprarenal,  i  c.c.  of  a  i  to  10,000  solution  of  adrenalin 
chlorid. 

The  effect  of  the  suprarenal  extract  upon  skeletal  muscle  has 
already  been  shown  under  Muscle-nerve  Physiology. 

4.  Remove  the  second  suprarenal  from  rabbit  i  as  follows: 
Place  the  animal  under  morphine  and  ether.  Tie,  belly  down, 
upon  the  operating-board,  placing  a  pad  under  the  abdomen  for 
the  purpose  of  bringing  the  viscera  into  the  field  of  the  operation. 
Shave  and  cleanse  the  skin  of  the  right  flank.  Beginning  at  the 
lower  border  of  the  ribs,  make  a  longitudinal  incision  through  the 
skin  and  fascia  of  the  flank  about  three  inches  long  and  about  two 
inches  from  the  spinous  processes  of  the  vertebrae.  Cut  through 
the  lumbar  aponeurosis  of  the  abdominal  muscles,  and  separate 
these  from  the  heavy  spinous  muscles.  Locate  the  kidney.  Let 
an  assistant,  standing  on  the  right  side  of  the  operating-table,  hold 
the  kidney  down  and  to  the  right  with  one  finger,  and  with  two  fin- 
gers of  the  other  hand  pull  the  lateral  margin  of  the  wound  up 
and  out.  The  operator  should  stand  to  the  left  of  the  subject. 
Follow  up  the  renal  vein  as  before,  until  the  right  suprarenal  body 
is  seen.  Separate  this  from  its  attachments  in  the  same  way  as 
was  done  in  removing  the  left  suprarenal.  Remove  the  gland  en- 
tire, or,  if  this  cannot  be  done  with  safety,  remove  as  much  as  pos- 
sible and  crush  the  remainder  with  forceps.  Sew  up  the  wound 
with  two  rows  of  sutures  and  return  the  rabbit  to  its  cage.  Since 
death  frequently  occurs  within  the  first  twelve  hours  following  com- 
plete removal  of  the  suprarenals,  the  operation  is  preferably  done 
in  the  morning,  so  that  the  animal  may  be  under  observation  all 
day.  If  the  operation  has  been  successfully  performed  with  the 
minimum  of  shock  and  hemorrhage,  the  animal  should  regain  con- 
sciousness. Make  careful  note  of  all  symptoms  from  the  time  of 
the  operation  until  death  occurs. 


INTERNAL  SECRETIONS. 

At  the  autopsy  examine  all  the  organs  and  make  careful  search 
for  accessory  suprarenals  and  for  any  stump  remaining  after  re- 
moval of  the  glands. 

Compare  the  symptomatology  of  the  rabbit  minus  suprarenals 
with  the  symptomatology  of  Addison's  disease  in  man. 


CHAPTER  VIII. 
RESPIRATION. 

I.  RESPIRATORY  MOVEMENTS  IN  MAN. 

1.  Direct  Observation. — Let  one  of  the  students  of  a  labora- 
tory group,  preferably  one  of  slender  build,  strip  to  the  waist. 
Let  him  sit  straight  on  a  stool  with  the  arms  and  hands  symmetri- 
cally placed  at  the  sides,  and  both  sides  of  the  chest  evenly  illumi- 
nated. 

(a)  Observe  first  whether  or  no  the  two  sides  of  the  chest  are  of 
equal  size  and  the  respiratory  movements  equally  prominent  on 
the  two  sides.    Note  the  increase  in  the  lateral  and  antero-posterior 
diameters  of  the  thorax  in  inspiration.    Note  the  movements  of 
the  abdominal  wall  in  inspiration  and  in  expiration.     Note  the 
changes  in  the  intercostal  spaces  during  the  respiratory  movements. 

What  muscles  seem  to  be  involved  in  quiet  inspiration  ?  in 
quiet  expiration  ?  In  which  part  of  the  thorax  is  the  lateral  diame- 
ter enlarged  most,  during  quiet  inspiration  ?  What  are  the  move- 
ments of  the  ribs  during  expiration  and  inspiration  ?  Demonstrate 
these  movements  on  the  skeleton  thorax. 

Remembering  the  attachments  of  the  external  and  internal  in- 
tercostal muscles,  demonstrate  their  action  on  the  skeleton  thorax 
by  the  use  of  heavy  elastic  bands  attached  to  the  ribs  in  the  same 
way  as  the  intercostals  are  attached. 

(b)  Now  let  the  subject  of  the  observation  make  a  forced  in- 
spiration, followed  by  a  forced  expiration.    Compare  this  with  the 
movements  of  quiet  respiration.    What  additional  muscles  are  in- 
volved in  inspiration  ?  in  expiration  ? 

(c)  By  means  of  some  form  of  pneumograph  or  stethograph 
record  the  respiratory  movements.     This  may  be  conveniently 

[148] 


RESPIRATION. 

done  by  strapping  to  the  chest  a  rubber  bulb  connected  with  a  re- 
cording tambour  whose  lever  is  allowed  to  write  on  a  medium  fast 
drum.  A  time  tracing  in  seconds  should  also  be  taken. 

First  record  the  respiratory  movements  while  the  subject  is  in 
the  recumbent  position.  Take  the  pulse  rate  at  the  same  time. 
Allow  the  subject  to  sit,  and  record  the  respiratory  movements 
again.  Repeat  the  record  with  the  subject  standing.  Compare 
the  rate  and  character  of  breathing,  together  with  the  pulse  rate, 
in  the  three  postures. 

Compare  the  inspiratory  phase  with  the  expiratory  phase.  What 
is  the  ratio  between  the  two  ?  Under  what  pathological  conditions 
may  this  ratio  be  disturbed  and  in  what  way  ?  What  is  the  rela- 
tion between  rate  of  heart-beat  and  respiratory  rate  ?  Under  what 
pathological  conditions  is  this  ratio  disturbed  ? 

(d)  Let  the  subject  take  some  form  of  exercise  for  a  few  min- 
utes, such  as  running  up  a  flight  of  stairs.     Record  respiratory 
movements  again.     Compare  with  other  tracings.     Observe  rate 
of  heart-beat. 

(e)  While  a  tracing  of  the  respiratory  movements  is  being  taken 
let  the  subject  take  several  swallows  of  water  in  rapid  succession. 
While  the  swallowing  is  going  on,  what  is  the  effect  on  the  respira- 
tory movements  ?    To  what  is  this  effect  due  ? 

2.  Respiratory  Sounds.  Auscultation. — In  order  to  distin- 
guish abnormal  respiratory  sounds  in  pathologic  pulmonary  con- 
ditions it  is  necessary  to  be  familiar  with  the  normal.  There  are 
normal  individual  variations  which  must  also  be  taken  into  ac- 
count. It  is  therefore  well  to  examine  a  number  of  normal  chests. 

(a)  Vesicular  Breathing. — With  a  stethoscope,  first  listen,  dur- 
ing quiet  respiration,  at  about  the  fifth  or  sixth  right  intercostal 
space.  A  sound  more  distinct  and  of  longer  duration  on  inspira- 
tion will  be  heard.  The  character  and  quality  of  this  sound  is 
hard  to  describe.  It  is  best  compared,  perhaps,  to  the  sound  made 
when  the  leaves  of  a  tree  are  stirred  by  a  light  breeze. 

What  is  the  ratio  of  inspiratory  sound  to  expiratory  sound? 
Compare  this  with  the  ratio  of  inspiration  to  expiration. 

[J49] 


LABORATORY  MANUAL  OF  PHYSIOLOGY. 

(b)  Bronchial  Breathing.  —  With  a  stethoscope  listen  to  the 
breathing  over  the  trachea  and  over  the  second  costal  cartilage  on 
the  right  side.    The  latter  position  is  at  the  bifurcation  of  the  right 
bronchus.    The  sounds  heard  here  are  due  to  the  passage  of  air 
through  a  tube  and  in  part  over  the  mouth  of  a  tube.    This  is  a 
fair  example  of  normal  bronchial  breathing.    Compare  with  ve- 
sicular breathing.    Should  bronchial  breathing  be  heard  where  you 
listened  for  vesicular  breathing  ?    What  change  in  the  lung  might 
give  rise  to  bronchial  breathing  ? 

(c)  Listen  over  the  right  lung  during  forced  respiration.    How 
do  the  respiratory  sounds  differ  from  those  of  quiet  respiration  ? 

(d)  Now  go  over  the  chest  systematically,  first  one  side  and  then 
the  other,  comparing  the  two.    Start  at  the  supraclavicular  space 
of  one  side,  then  listen  in  the  same  region  on  the  other  side,  and  so 
on  over  all  of  the  anterior,  lateral,  and  posterior  aspects  of  the 
thorax.    Note  any  differences  in  the  sounds  in  the  different  regions 
of  the  chest  either  in  intensity,  or  duration,  or  character.    Explain. 

3.  Palpation.    Vocal  Fremitus. — Place  the  same  hand  suc- 
cessively over  the  various  regions  of  the  chest  both  in  front  and 
behind,  alternating  sides,  so  as  to  compare  one  region  with  its  fel- 
low of  the  opposite  side.    While  the  hand  is  applied  to  the  chest 
wall  let  the  subject  say  "  twenty-one  "  or  some  other  number.    The 
vibrations  of  the  chest  wall  during  phonation  are  distinctly  felt  by 
the  hand.    There  are  pathologic  variations  of  this,  either  in  an  ac- 
centuation of  the  fremitus  on  one  side  as  compared  with  the  other, 
or  in  a  diminution  of  the  fremitus. 

Listen  again  over  the  various  regions  of  the  chest  while  the  sub- 
ject counts  "one,  two,  three."  The  sound  will  be  transmitted  to 
the  ear,  but  more  as  a  murmur,  and  not  as  distinctly  enunciated 
syllables.  In  certain  pathologic  conditions  the  spoken  or  whis- 
pered word  is  very  distinctly  heard,  as  if  it  were  spoken  directly 
into  the  end  of  the  stethoscope.  What  physical  changes  in  the 
lung  might  cause  this  condition  ? 

4.  Percussion. — Laying  the  middle  finger  of  the  left  hand  be- 
tween the  ribs,  in  the  intercostal  spaces,  and  using  the  bent  mid- 


RESPIRATION. 

die  finder  of  the  right  hand  as  a  hammer,  percuss  the  entire  chest 
wall.  Explain  the  variations  in  the  percussion  note  over  various 
regions.  Map  out  the  heart  and  the  liver. 

6.  Chest  Measurements.  —  (a)  Let  a  student  strip  to  the 
waist.  Measure  the  chest  circumference  at  the  axillae  at  the  end 
of  quiet  inspiration  and  at  the  end  of  quiet  expiration.  Repeat 
this  measurement  at  the  level  of  the  end  of  the  sternum.  Re- 
peat both  measurements,  in  forced  inspiration  and  expiration. 
Record  results. 

(b)  In  the  same  individual  measure  the  antero-posterior  tho- 
racic diameters  at  the  junction  of  the  first  and  second  pieces  of  the 
sternum  and  at  the  end  of  the  sternum,  during  inspiration  and  ex- 
piration, both  quiet  and  forced.  Measure  the  changes  in  the  lat- 
eral diameters  in  the  same  way.  A  pair  of  long,  graduated  calipers 
serves  for  the  purpose  of  making  the  measurements.  Record  results. 

For  purposes  of  comparison,  measure  the  length  of  the  trunk 
from  the  "vertebra  prominens"  to  the  level  of  the  chair  upon 
which  the  subject  is  sitting.  Repeat  these  measurements  on  other 
students  and  keep  a  record  for  each  individual. 

6.  Respiratory  Capacity.  —  This  is  usually  determined  by 
some  form  of  water  spirometer.  A  long,  narrow  cylinder,  gradu- 
ated in  cubic  centimetres,  is  filled  with  water  and  inverted  over 
water  in  another  cylinder.  An  air  tube  passes  through  the  second 
cylinder  to  the  top  of  the  first.  The  first  cylinder  is  counterpoised 
by  weights  and  pulleys,  so  that  when  air  is  forced  into  it  the  water 
is  displaced,  the  cylinder  rises,  and  the  amount  of  water  displaced 
by  air  can  be  read  off  on  the  attached  scale. 

(a)  Calibration  of  Spirometer. — The  air  cylinder  of  the  spirom- 
eter should  be  calibrated  before  using.  This  may  conveniently 
be  done  in  the  following  way:  Fill  the  cylinder  with  air;  note  the 
position  of  the  pointer  on  the  scale;  place  the  outlet  air  tube  under 
a  graduated  1000  c.c.  cylinder,  filled  with  water  and  inverted  over 
water,  in  a  large  pan  or  tub ;  slowly  depress  the  spirometer  cylin- 
der until  the  pointer  has  passed  five  or  ten  spaces  of  the  scale,  and 
read  off  the  amount  of  water  displaced  from  the  graduated  cylin- 


LABORATORY  MANUAL  OF  PHYSIOLOGY. 

der  in  the  tub",  record  and  repeat  the  operation  until  all  the  spirom- 
eter  scale  is  estimated  in  terms  of  cubic  centimetres  of  the  testing 
graduated  cylinder. 

(b)  Tidal  Air. — With  the  pointer  of  the  spirometer  at  the  zero 
mark  of  the  scale,  expire  into  the  spirometer  cylinder  after  an  or- 
dinary inspiration.    The  amount  of  expired  air  recorded  is  an  ap- 
proximate indication  of  the  tidal  air,  i.e.,  the  amount  that  passes 
in  and  out  of  the  lungs  during  quiet  respiration. 

(c)  Supplemental  Air. — Repeat  the  spirometer  record,  taking  a 
normal  quiet  inspiration,  and  then  forcing  as  much  air  out  of  the 
lungs  as  possible.    Read  the  record  on  the  spirometer  scale.    Sub- 
tract the  reading  of  (b)  from'  the  latter  reading.    The  difference  is 
the  so-called  supplemental  or  reserve  air.    This  is  the  amount  of 
air  which  remains  in  the  lung  after  a  quiet  expiration  and  which 
may  be  expelled  by  a  forced  expiration.    After  this  is  expelled,  air 
still  remains  in  the  lung  which  cannot  be  forced  out.    This  is  the 
residual  air.    The  air  which  can  be  inspired  in  addition  to  the  or- 
dinary inspiration  is  known  as  the  complemental  air. 

(d)  Vital  Capacity. — Take  the  deepest  possible  inspiration,  and 
empty  the  lungs  as  completely  as  possible  into  the  spirometer 
cylinder  by  a  forced  expiration.    The  record  obtained  indicates 
the  full  pulmonary  capacity  minus  the  residual  air,  and  is  equal 
to  the  sum  of  the  tidal  air,  complemental  air,  and  supplemental  air. 
This  is  known  as  the  vital  capacity. 

Record  the  vital  capacity  of  each  member  of  your  group,  and 
compare  with  the  various  chest  measurements  already  taken. 

7.  Cardio-pneumatic  Movements. — The  changes  in  volume  of 
the  heart  during  systole  and  diastole  cause  corresponding  changes 
in  the  capacity  of  the  thorax  and  consequently  of  the  lungs.  The 
inspiratory  and  expiratory  movements  caused  by  the  heart-beat 
may  be  demonstrated  in  the  following  manner:  Bend  one  end  of 
a  medium  large  piece  of  glass  tubing  into  the  form  of  a  U.  Fill  the 
bend  of  the  U  with  a  little  water  colored  with  eosin.  Place  the  end 
of  the  horizontal  limb  of  this  tube  in  one  nostril.  Close  the  other 
nostril  with  the  finger  and  keep  the  mouth  closed.  Hold  the 


RESPIRATION. 

breath.  The  fluid  in  the  U  will  move  synchronously  with  the 
heart-beat.  At  each  systole  there  will  be  an  inspiratory  move- 
ment, and  at  each  diastole  an  expiratory  movement. 

II.  PULMONARY  PRESSURE. 

Place  a  rabbit  under  morphine  narcosis.  Anaesthetize  lightly 
with  ether.  Place,  back  down,  on  rabbit-board.  Expose  the 
trachea  through  a  median  cervical  incision.  Introduce  a  tracheal 
cannula.  Connect  this,  through  a  T-tube,  with  the  proximal  end  of 
a  mercury  manometer.  Place  a  clip  on  the  rubber  tubing,  leading 
from  the  T  to  the  manometer,  so  that  it  can  be  either  opened  or 
closed.  At  the  beginning  of  an  inspiration,  open  the  manometer 
clip  and  close  the  air-inlet  limb  of  the  T.  At  the  height  of  inspira- 
tion, close  the  clip  on  the  manometer  tube  and  open  the  air-inlet 
tube.  The  inspiratory  negative  pressure  may  then  be  read  off  from 
the  manometer  scale.  A  tracing  of  the  respiratory  movements 
may  be  obtained  in  this  way  by  partially  occluding  the  air-inlet 
tube.  Close  the  air-inlet  tube  during  several  respiratory  cycles. 
Note  the  positive  pressure  of  the  expiratory  phase  and  the  negative 
pressure  of  the  inspiratory  phase.  After  a  few  respiratory  efforts 
the  animal  will  begin  to  struggle  because  of  asphyxia.  When  this 
occurs,  note  the  change  in  the  character  of  the  respirations  and  the 
great  difference  between  inspiratory  and  expiratory  pressure.  In 
quiet  respiration,  is  the  positive  pressure  in  expiration  much  above 
the  atmospheric  pressure  ? 

III.  INTRATHORACIC  PRESSURE. 

Using  the  same  rabbit  as  in  the  previous  experiment,  connect  the 
proximal  limb  of  a  manometer,  through  a  piece  of  pressure  tubing, 
with  a  small  glass  tube.  Make  a  small  incision  through  the  skin 
over  the  fourth  intercostal  space  on  the  right  side.  Make  a  very 
small  nick  through  the  intercostal  muscles  and  force  the  glass  tube 
through  this  opening.  The  tube  should  fit  so  tightly  that  there 
will  be  no  leakage  of  air  around  it.  Note  the  movements  of  the 
mercury  in  the  manometer  with  inspiration  and  expiration.  Com- 


LABORATORY  MANUAL  OF  PHYSIOLOGY. 

pare  them  with  the  changes  of  intrapulmonary  pressure.  In  quiet 
breathing,  what  is  the  constant  condition  of  pressure  in  the  thorax  ? 
How  does  this  pressure  change  with  inspiration  and  expiration? 
Explain. 

Now  close  the  tracheal  cannula  and  induce  asphyxia.  Note  the 
variations  in  intrathoracic  pressure  during  the  violent  respiratory 
efforts  which  occur  in  this  condition. 

For  the  influence  of  the  respiratory  movements  on  the  blood 
pressure  and  heart-beat,  refer  back  to  your  blood-pressure  tracings, 
and  study  again  the  respiratory  waves  of  the  tracings.  When  is 
the  intrathoracic  pressure  lowest?  when  highest?  What  effect 
would  changes  of  pressure  within  the  thorax  have  upon  the  pressure 
of  blood  in  the  large  arteries  and  veins  and  in  the  heart  itself  dur- 
ing diastole  ? 

IV.  THE  VAGUS  NERVE  IN  RESPIRATION. 

The  same  rabbit  that  was  used  in  the  previous  experiments  may 
be  used  for  this.  Continue  the  dissection  of  the  neck  region  very 
carefully,  isolating  both  vagus  nerves,  and  both  superior  laryn- 


FIG.  37.— To  Record  Movements  of  Diaphragm.    JR,  Recording  tambour ;  B,  rubber 
bulb,  connected  with  recording  tambour ;  D,  diaphragm ;  L,  liver. 

geals.  Make  an  incision  through  the  abdominal  wall  below  the 
xyphoid  appendix  of  the  sternum,  just  large  enough  for  the  intro- 
duction of  a  catheter  over  the  end  of  which  a  collapsed  rubber  bal- 
loon is  tied.  Pass  this  up  between  the  diaphragm  and  the  liver,  on 


RESPIRATION. 

the  right  side.  Connect  the  catheter  with  the  recording  tambour 
(see  Fig.  37),  and  the  movements  of  the  diaphragm  will  be  recorded 
upon  a  drum  revolving  at  medium  speed. 

(a)  First  take  a  tracing  of  the  normal  movements  of  the  dia- 
phragm.   Take  a  time  tracing  in  conjunction  with  the  respiratory 
tracing.    Note  the  rate  of  the  respirations  in  the  rabbit.    These 
may  be  influenced  by  the  morphine  which  has  been  previously 
given  the  rabbit. 

(b)  While  the  tracing  is  being  taken,  tickle  the  rabbit's  nose 
with  a  feather.    Is  there  any  effect  on  the  respiratory  movements  ? 
What  nerve  has  been  stimulated  ? 

Note  the  movements  of  the  rabbit's  nostrils  with  inspiration  and 
expiration.  These  movements  continue,  even  though  breathing  is 
no  longer  taking  place  through  the  nose,  but  through  the  tracheal 
cannula.  Other  associated  movements  you  have  already  noted, 
e.g.,  the  opening  and  closing  of  the  glottis. 

(c)  Pinch  the  skin  or  pour  a  little  ether  on  the  shaved  abdom- 
inal surface.    What  effect  has  cutaneous  stimulation  on  respiratory 
movements  ? 

(d)  Now  tie  one  vagus  with  two  ligatures  and  cut  between. 
Mark  on  the  tracing  the  time  at  which  the  vagus  was  tied  and  cut. 
Note  any  change  in  the  respiratory  movements  occurring  at  the 
time  of  tying  and  cutting  the  nerve  or  following  this  operation.    If 
the  respiratory  rhythm  has  been  disturbed,  does  it  return  to  nor- 
mal, after  a  time  ? 

(e)  Allow  "five  or  ten  minutes  to  elapse  and  then  stimulate  the 
central  end  of  the  cut  vagus  with  a  weak  tetanizing  current.    What 
is  the  effect  on  the  respiratory  movements  ? 

(/)  Stimulate  the  central  end  of  the  cut  vagus  with  a  medium 
strong  tetanizing  current.  Compare  this  effect  with  that  obtained 
with  the  weak  stimulation. 

(g)  Apply  to  the  central  end  of  the  cut  nerve  a  few  crystals  of 
NaCl.  Note  the  change  in  the  respiratory  movements.  After  the 
effect  of  this  stimulus  is  sufficiently  evident,  cut  off  the  small  piece 
of  nerve  to  which  the  salt  has  been  applied. 


LABORATORY  MANUAL  OF  PHYSIOLOGY. 

(h)  Stimulate  one  superior  laryngeal  nerve  with  a  weak  tetaniz- 
ing  current.  Note  the  effect  on  the  respiratory  movements.  Stim- 
ulate with  a  stronger  current  and  note  the  effect. 

(i)  Now  tie  and  cut  the  other  vagus  nerve.  Both  vagi  are  now 
severed.  What  is  the  effect  on  the  rhythm,  rate,  and  depth  of  the 
respiratory  movements  ? 

(y)  Stimulate  both  central  ends  of  the  cut  nerves  with  a  weak 
tetanizing  current.  Note  the  effect  on  the  rate,  rhythm,  and  char- 
acter of  the  respirations.  Stimulate  with  a  stronger  current. 
Result? 

(k)  Stimulate  both  vagi  with  a  weak  tetanizing  current,  and, 
when  the  effect  of  such  stimulation  begins  to  show,  stimulate 
both  superior  laryngeal  nerves  with  a  medium  strong  current. 
Result  ? 

In  all  of  the  above  experiments,  marks  should  be  made  upon 
the  tracings  to  indicate  the  operative  procedures  and  their  time 
relation  to  the  tracing;  the  nerves  stimulated  and  the  strength  and 
nature  of  the  stimulus  employed. 

(/)  Stimulate  the  peripheral  ends  of  the  divided  vagus  nerves. 
Is  there  any  effect  on  the  respiratory  movements  ? 

From  the  above  experiments  what  conclusions  can  you  draw 
concerning  the  function  of  the  vagus  nerve  in  relation  to  respira- 
tion ?  Is  the  vagus  chiefly  an  afferent  or  an  efferent  nerve  in  re- 
lation to  the  lungs? 

The  vagus  has  now  been  studied,  in  part,  in  connection  with  the 
circulation,  digestion,  and  respiration.  Compare  its  functions  in 
relation  to  the  three  systems.  It  contains  both  afferent  and  effer- 
ent fibres  for  circulation,  digestive  tract,  and  respiratory  tract. 
Summarize  your  knowledge  on  the  subject,  secured  in  part  through 
your  own  experiments  in  the  laboratory. 

V.  INNERVATION  OF  THE  DIAPHRAGM. 

i.  Still  using  the  same  rabbit  that  was  used  in  the  previous  ex- 
periments, expose  the  phrenic  nerve  of  the  right  side.  This  may 
be  done  in  the  following  manner:  Enlarge  the  cervical  incision  to 


RESPIRATION. 

the  upper  end  of  the  sternum.  Pull  the  sterno-mastoid  and  other 
longitudinal  neck  muscles  toward  the  median  line.  Pull  the  skin 
and  other  muscles  to  one  side  and  expose  the  cervical  spinal  nerves 
and -the  beginning  of  the  brachial  plexus.  The  carotid  artery, 
vagus  nerve,  and  jugular  vein  should  also  be  pulled  toward  the 
median  line.  Determine  the  position  of  the  fourth,  fifth,  sixth, 
and  seventh  cervical  nerves.  Arising  by  filaments  from  the  pos- 
terior divisions  of  these  nerves,  a  fine  nerve  fibre  will  be  seen  run- 
ning over  the  heavy  spinous  muscles,  parallel  with  the  spinal  col- 
umn and  disappearing  under  the  clavicle.  To  make  sure  that  you 
have  found  the  phrenic  nerve,  place  fine  platinum  electrodes  under 
this  nerve  filament  and  stimulate  with  medium  strong  single-in- 
duction shocks.  The  diaphragm  recorder  which  records  the  move- 
ments of  the  right  side  of  the  diaphragm  will  move  with  each 
stimulus. 

2.  Pass  a  thread  around  the  upper  origin  of  the  nerve.    Tie  the 
nerve  and  sever  all  connection  with  the  cervical  nerves.    Note  the 
diminution  or  complete  loss  of  recorded  movement  of  the  dia- 
phragm.   The  right  side  of  the  diaphragm  is  paralyzed  and  moves 
only  as  it  is  pulled  upon  by  the  side  which  is  still  active.    Compare 
the  thoracic  breathing  after  the  section  of  one  phrenic  with  the 
thoracic  breathing  before  the  section  of  the  nerve. 

Do  the  two  sides  of  the  chest  move  equally,  or  is  there  a  differ- 
ence between  the  right  and  the  left  side  ? 

3.  Stimulate  the  nerve  with  single  shocks  from  an  inductorium, 
and  take  a  tracing  on  the  drum. 

4.  Enlarge  the  abdominal  opening  and  pull  down  the  abdom- 
inal viscera  so  that  the  movements  of  the  diaphragm  may  be  ob- 
served directly.    Note  the  movement  of  the  left  side,  whose  nerve 
supply  is  still  intact.    Note  the  lack  of  motion  on  the  right  side  and 
the  position  of  this  enervated  side  of  the  diaphragm  during  in- 
spiration and  expiration. 

5.  Expose  and  cut  the  left  phrenic  nerve.     Both  sides  of  the 
diaphragm  are  now  paralzyed.    Note  the  change  in  the  respira- 
tory movements.    If  the  rabbit  is  young  there  will  be  great  difn- 

[157] 


LABORATORY  MANUAL  OF  PHYSIOLOGY. 

culty  in  breathing,  amounting  to  distinct  dyspnoea.    Note  the  in- 
crease in  thoracic  breathing. 

Explain  the  difficulty  in  breathing  following  the  paralysis  of  the 
diaphragm.  Is  it  due  simply  to  the  loss  of  the  active  movements 
of  the  diaphragm  in  enlarging  the  vertical  diameter  of  the  thorax? 

6.  Place  both  phrenic  nerves  on  electrodes  from  the  inducto- 
rium.    Stimulate  with  a  medium  strong  tetanizing  current,  inter- 
rupted about  thirty  times  per  minute.     Is  the  dyspncea  relieved  ? 
Does  the  rabbit  cease  attempts  at  thoracic  breathing  ? 

7.  Close  the  trachea  by  means  of  an  artery  clamp  and  kill  the 
rabbit  by  asphyxiation.    Note  all  phenomena  connected  with  death 
from  this  cause.    After  breathing  has  ceased,  open  the  thorax  and 
observe  the  heart.    Is  this  still  beating?    Note  the  color  of  the 
blood.    Note  difference  between  the  two  sides  of  the  heart.    Excise 
the  heart.    Does  the  heart-beat  recover  for  a  short  time  after  ex- 
cision ? 

VI.  EFFECT  OF  BLOOD  TEMPERATURE  ON  RESPIRATION. 

1.  Heat. — Narcotize  a  rabbit.  Place,  back  down,  on  the  rabbit- 
board.  Through  a  median  cervical  incision  expose  both  carotid 
arteries  and  isolate  the  vagus  nerves.  Arrange  the  apparatus  for 
recording  the  movements  of  the  diaphragm.  Isolate  as  much  of 
each  carotid  as  possible.  Separate  the  artery  from  the  nerves 
running  with  it,  by  several  layers  of  paper.  Tie  each  carotid, 
gently,  to  a  small  tubing  running  parallel  to  the  artery.  One  end 
of  this  tubing  is  connected  to  a  rubber  outlet  tube.  The  other  end 
is  connected  to  a  rubber  inlet  tube.  The  inlet  tube  leads  from  a 
bottle  filled  with  water  kept  at  a  temperature  of  40  C.,  by  immer- 
sion in  a  water-bath.  This  bottle  is  elevated  sufficiently  to  give  a 
constant  flow  of  warm  water  through  the  tubes  in  contact  with  the 
arteries.  The  blood  passing  through  the  carotids  is  therefore 
warmed  two  or  three  degrees  above  the  normal. 

Set  up  the  arrangement  as  above  described.  First  take  a  normal 
respiratory  tracing.  Then,  while  a  tracing  is  being  taken,  let  the 
warm  water  run  through  the  tubing  and  note  the  effect  upon  res- 


RESPIRATION. 

piration.  Change  in  the  pulse  rate  may  also  be  observed  by  watch- 
ing the  pulsation  of  the  carotid  arteries  or  by  feeling  the  cardiac 
impulse  on  the  chest  wall.  How  are  the  respirations  affected  when 
the  blood  going  to  the  brain  is  warmed  above  the  normal?  How 
is  the  heart-beat  affected?  What  is  the  relation  between  heart- 
beat, respiratory  rate,  and  rise  of  temperature  ? 

2.  Cold. — Disconnect  the  warm-water  bottle  from  the  tubes  in 
contact  with  the  carotids,  and  substitute  a  receptacle  containing  ice 
water.  First,  let  the  respiratory  rate  return  to  normal.  Then 
while  a  tracing  is  being  taken,  allow  the  cold  water  to  flow  through 
the  tubing  in  contact  with  the  arteries.  Note  the  effect  upon 
the  respiration  and  the  heart-beat.  Compare  the  tracing  obtained 
through  the  cold  application  with  that  obtained  when  heat  was 
applied. 

What  is  the  effect  of  warmed  blood  upon  the  respiratory  centre  ? 
of  cold  blood  upon  the  respiratory  centre  ? 

VII.  EFFECT  OF  ANEMIA  UPON  THE  RESPIRATORY  CENTRE. 

1.  Using  the  same  animal  as  in  the  previous  experiments,  clamp 
both  carotids  with  artery  clips.    Is  there  any  change  in  respiration 
following  the  occlusion  of  both  carotids  ?    If  so,  does  this  changed 
respiration  continue  or  does  it  soon  return  to  the  normal?    Ex- 
plain.    Does  occlusion  of  the  carotids  materially  or  permanently 
diminish  the  blood  supply  to  the  brain?    What  is  the  collateral 
circulation  ? 

What  would  be  the  only  way  completely  to  cut  off  the  blood 
supply  to  the  brain  ? 

2.  Tie  both  carotids  as  high  up  as  practicable.     Introduce  a 
glass  cannula  into  each  artery.     Take  a  normal  respiratory  trac- 
ing.   While  this  is  being  taken,  open  the  clips  on  both  arteries  and 
allow  the  animal  to  bleed  to  death.    Collect  the  blood  in  a  grad- 
uated cylinder  and  record  the  phenomena  observed  after  the  loss 
of  each  additional  10  c.c.  of  blood.    How  much  blood  is  lost  before 
the  respirations  are  affected?    In  what  way  are  the  respirations 
affected  as  hemorrhage  continues  ? 

[159] 


LABORATORY  MANUAL  OF  PHYSIOLOGY. 

Note  also  every  few  seconds  the  condition  of  the  various  re- 
flexes. Among  these  try  the  reaction  of  the  pupil  to  light,  the  con- 
junctival  reflex,  the  reflex  upon  tickling  the  nares,  and  cutaneous 
reflexes.  Note  the  time  at  which  each  one  of  these  reflexes  disap- 
pears. Note  time  when  struggling  begins.  How  do  these  respira- 
tory phenomena  differ  from,  and  resemble,  those  of  asphyxia  ? 

VIII.  RESPIRATORY  CENTRE. 

Narcotize  a  rabbit  lightly  with  morphine.  Anaesthetize  with 
ether.  Expose  the  trachea  and  both  carotid  arteries  through  a 
median  cervical  incision.  Introduce  a  cannula  into  the  trachea 
and  tie  both  carotids. 

Change  the  position  of  the  animal  so  that  it  lies  on  the  rabbit- 
board  belly  down.  Make  an  incision  in  the  median  line  through 
the  integument  of  the  skull  from  the  root  of  the  nose  to  the  occiput. 
Pull  the  skin  flaps  to  one  side,  exposing  the  parietal  bones  of  the 
skull.  Make  two  trephine  openings,  one  through  the  parietal  bone 
of  each  side,  enlarging  the  openings  with  cutting  forceps,  until  the 
entire  skull  cap  is  removed.  Be  careful  in  crossing  the  median 
line  not  to  injure  the  longitudinal  venous  sinus. 

1 .  Open  and  lay  back  the  dura  on  each  side,  thus  exposing  both 
cerebral    hemispheres.     With  a  blunt  spade  or  scalpel   handle 
crush  both  cerebral  lobes.     Control  the  hemorrhage  by  packing 
with  cotton  moistened  with  adrenalin  i  to  10,000,  or  use  the  actual 
cautery. 

Observe  the  respiratory  movements  before,  during,  and  after 
this  operation.  Do  respirations  continue  after  the  removal  of  the 
cerebrum?  Is  the  controlling  respiratory  centre  located,  there- 
fore, in  the  excised  portion  of  brain  ? 

2.  Continue  the  median  dorsal  incision  until  all  the  cervical  ver- 
tebrae are  exposed.     Continue  the  removal  of  the  skull  cap  until 
the  cerebellar  hemispheres  are  exposed.    Remove  the  cerebellar 
hemispheres  in  the  same  way  that  the  cerebral  lobes  were  re- 
moved. 

Do  the  respiratory  movements  cease  in  the  absence  of  the  cere- 

[160] 


RESPIRATION. 

bellum?    Is  the  controlling  respiratory  centre,  therefore,  located 
in  the  cerebellum  ? 

3.  Divide  the  upper  part  of  the  medulla  by  an  incision  between 
the  atlas  and  skull.    Do  the  respiratory  movements  continue  ? 

4.  Divide  the  cord  on  a  level  with  the  origin  of  the  seventh  cer- 
vical nerve.    In  the  rabbit  the  respirations  are  altered  but  little, 
since  in  this  animal  breathing  is  chiefly  diaphragmatic.    By  this 
section  the  thoracic  muscles  are  cut  off  from  the  respiratory  centre, 
but  the  innervation  of  the  diaphragm  is  still  intact.    Impulses  are 
still  carried  to  the  centre  from  the  periphery  by  the  intact  vagus 
nerves.    Other  afferent  impulses  are  in  the  main  cut  off  from  the 
centre  by  its  isolation  from  the  brain  above  and  the  cord  below. 

5.  Expose  and  cut  both  vagus  nerves  in  the  neck  region.    Note 
the  change  in  the  character  of  the  respiratory  movements  and  the 
disturbance  of  the  respiratory  rhythm.    Are  the  respiratory  move- 
ments which  still  continue   sufficient   to   sustain  the  life  of   the 
animal  ? 

Allow  the  first  stages  of  asphyxia  to  occur.    Then  revive  the  an- 
imal with  artificial  respiration  continued  for  a  short  time. 

6.  Place  the  central  ends  of  the  divided  vagi  on  electrodes  from 
an  inductorium  set  up  for   medium   strong  tetanizing  currents. 
Stimulate  the  vagi  with  this  current,  interrupted  about  thirty  times 
per  minute.     Stop  the  artificial  respiration.     Is  the  respiratory 
rhythm  re-established  ?    Compare  the  results  obtained  from  this 
experiment  with  those  obtained  through  section  and  stimulation 
of  the  vagi  in  former  experiments.     What  conclusions  can  you 
draw  concerning  the  location  of  the  respiratory  centre  and   the 
regulation  of  its  rhythmic  activity  ? 

IX.    CONDITION  OF  LUNG  FOLLOWING  SECTION  OF  BOTH  VAGI. 

Narcotize  and  anaesthetize  a  rabbit.  Under  aseptic  precautions 
expose  and  cut  both  vagus  nerves  in  the  neck  region.  Sew  up  the 
wound  and  return  the  animal  to  its  cage.  Make  careful  observa- 
tions of  the  subject  until  death  occurs,  which  will  generally  be 
within  forty-eight  hours.  Determine  the  cause  of  death  at  au- 
ii  [161] 


LABORATORY  MANUAL  OF  PHYSIOLOGY. 

topsy.  Observe  particularly  the  condition  of  the  lungs.  Save 
pieces  of  the  lung  tissue  for  hardening,  embedding,  and  sectioning. 
Stain  sections  with  haematoxylin  and  eosin  and  study  with  the 
microscope. 

What  pulmonary  condition  follows  section  of  the  vagus  nerves 
and  how  is  it  brought  about  ?  Save  these  sections  for  comparison 
with  sections  of  human  lung  showing  areas  of  lobar  and  lobular 
pneumonia. 

X.  ARTIFICIAL  RESPIRATION. 

The  student  should  be  familiar  with  at  least  one  good  nethod 
of  artificial  respiration  for  use  in  emergencies.  One  of  the  best 
methods  for  this  purpose  is  the  so-called  Sylvester's  method. 

It  consists  in  imitating,  so  far  as  possible,  the  normal  respiratory 
movements.  In  applying  this  method  the  operator  should  assure 
himself  that  the  respiratory  passages  of  the  subject  are  free.  The 
subject  is  placed  upon  his  back,  the  shoulders  being  elevated  by 
some  support  placed  beneath  them.  The  head  should  be  on  a 
lower  level  than  the  feet. 

The  operator  should  stand  at  the  head  of  the  subject  and,  grasp- 
ing the  wrists,  flex  the  forearm  upon  the  arm  and  press  both  arms 
firmly  against  the  sides  of  the  chest,  pressing  down  and  in  on  the 
chest  at  the  same  time.  This  motion  forces  air  out  of  the  lungs. 
When  the  pressure  upon  the  chest  is  released,  the  thorax  through 
its  own  elasticity  rebounds  to  its  original  capacity,  and  air,  by  this 
motion  alone,  is  drawn  into  the  lungs.  The  thoracic  diameters  are 
still  further  increased  by  the  second  part  of  the  operation. 

This  consists  of  extending  the  arms  and  pulling  them  above  the 
head,  giving  an  extra  tug  when  the  position  of  full  extension  has 
been  reached.  The  accessory  respiratory  muscles,  mainly  the 
pectorals,  are  thus  put  on  the  stretch  and  in  their  turn  pull  up 
and  out  on  the  upper  part  of  the  thorax. 

After  this  has  been  accomplished,  the  first  position  is  again  as- 
sumed and  expiration  is  brought  about.  This  alternate  forced' ex- 
piration and  inspiration  are  continued  at  the  rate  of  fifteen  to  twenty 


RESPIRATION. 


per  minute,  until  the  subject  begins  to  make  respiratory  efforts  of 
his  own  or  no  doubt  remains  that  life  is  extinct. 

XI.  ESTIMATION  OF  CO2  AND  H2O  EXPIRED  IN  A  GIVEN  TIME. 

The  estimation  of  carbon  dioxid  and  water  expired  by  a  small 
animal  in  a  given  time  may  be  conveniently  done  by  each  group 
of  students.  Absorption  tubes,  made  for  the  purpose,  may  be 


FIG.  38.— Apparatus  for  Estimating  the  Water  and  Carbon  Dioxid  Eliminated  from 
the  Lungs  of  a  Small  Animal.    Described  in  text. 

bought  at  a  moderate  price,  as  well  as  the  respiratory  chamber 
with  air  inlet  and  outlet  and  tightly  fitting  stopper.  A  schematic 
sketch  of  the  apparatus  used  is  given  in  Fig.  38. 

The  chamber  C  is  intended  for  holding  some  small  animal,  such 
as  a  rat  or  a  mouse.  This  is  tightly  stoppered,  the  stopper  being 
perforated  for  the  passage  of  two  tubes — (a)  the  air-intake  tube, 
which  passes  nearly  to  the  bottom  of  the  chamber,  and  (b)  the  air- 
outlet  tube,  which  begins  near  the  upper  part  of  the  chamber.  The 
air-intake  tube  is  connected  with  two  absorption  tubes — i ,  contain- 
ing pumice  stone  soaked  in  sulphuric  acid,  for  absorbing  the  moist- 
ure from  the  air  which  passes  to  the  respiratory  chamber,  and  2, 
containing  soda  lime  for  absorbing  the  carbon  dioxid  of  the  in- 
spired air. 

The  air  outlet  tube  is  also  connected  with  two  absorption  tubes — 
3,  containing  sulphuric  acid  for  absorbing  the  water  of  the  expired 
air,  and  4,  containing  a  strong  solution  of  sodium  hydrate  for  ab- 


LABORATORY  MANUAL  OF  PHYSIOLOGY. 

sorbing  the  carbon  dioxid  of  the  expired  air.    This  tube  also  has 
a  bulb  containing  CaCl2. 

Tube  4  is  connected  with  the  water  pump  P.  Air  is  drawn 
through  the  system  of  tubes  and  respiratory  chamber  in  the  di- 
rection of  the  arrow. 

1.  Set  up  the  apparatus  as  described,  and  allow  the  air  to  be 
drawn  through  for  fifteen  or  twenty  minutes  or  until  the  respira- 
tory chamber  has  been  freed  from  moisture  and  carbon  dioxid. 
Now  place  the  mouse  in  the  chamber,  stopper  quickly,  and  weigh. 
Also  quickly  weigh  tubes  3  and  4.    Make  a  record  of  the  weights 
for  use  later.    Connect  up  the  absorption  tubes  with  the  chamber 
and  air  pump,  and  allow  a  current  of  air  to  pass  through  the  system 
for  twenty  minutes  to  one-half  hour.    Then  disconnect  and  weigh 
the  chamber  and  absorption  tubes  again. 

The  difference  between  the  two  weighings  of  the  respiratory 
chamber  indicate  the  loss,  in  part,  of  the  animal,  in  water  and  CO2. 
The  difference  between  the  weighings  of  the  absorption  tubes  in- 
dicates their  gain  in  water  and  CO2,  respectively,  and  the  actual 
output  of  the  animal  in  carbon  dioxide  and  water,  during  the 
time  of  the  experiment.  The  difference  between  the  two  weigh- 
ings of  the  respiratory  chamber  and  the  difference  between 
the  two  weighings  of  the  absorption  tubes  do  not  correspond, 
since  what  the  animal  has  lost  in  CO2  it  has  partly  regained  in 
oxygen. 

The  gain  in  oxygen  may  be  roughly  determined  by  subtracting 
the  difference  between  weighings  of  chamber  C  from  the  difference 
between  weighings  of  tube  4.  The  ratio  between  the  oxygen  ab- 
sorbed and  the  carbon  dioxid  expired  is  known  as  the  respiratory 
quotient. 

What  is  the  respiratory  quotient  for  the  mouse  experimented 
upon? 

2.  Remove  the  mouse  from  the  respiratory  chamber.    Ventilate 
the  chamber  again.    Prepare  and  weigh  a  new  set  of  absorption 
tubes.    Give  the  mouse  some  form  of  exercise,  such  as  moving  the 
treadwheel  of  a  squirrel  cage.    Place  in  the  chamber  and  quickly 

[164] 


RESPIRATION. 

weigh.  Determine  the  CO2  elimination  for  another  period  equal 
to  the  first  period  in  time. 

How  does  the  CO2  elimination  of  the  second  period  compare 
with  that  of  the  first?  What  is  the  difference  in  the  respiratory 
quotient  ? 

Repeat  these  experiments,  using  a  cold-blooded  animal  instead 
of  the  mouse.  The  frog  will  serve  as  a  type.  The  mouse  is  an 
animal  of  high  respiratory  activity.  The  frog  is  an  animal  of  low 
respiratory  activity.  What  is  the  reason  for  a  higher  rate  of  gas 
exchange  in  the  mouse  than  in  the  frog  ? 

Study  the  nature  of  the  respiratory  movements  hi  the  frog.  How 
do  they  differ  in  mechanism  from  those  of  mammalia?  How  is 
respiration  carried  on  in  fishes  and  in  gilled  amphibia  ? 

What  points  in  common  have  these  three  orders  of  animals  as 
far  as  respiration  is  concerned  ?  What  points  of  difference  ? 

A  fair  idea  of  the  function  of  the  circulation  in  respiration  may 
be  obtained  from  a  study  of  the  circulation  through  the  gill  of 
Necturus. 


CHAPTER  IX. 

EXCRETION. 

IT  is  assumed  that  the  chemical  examination  of  the  urine,  both 
for  normal  and  abnormal  constituents,  has  already  been  done  by 
the  student  under  the  direction  of  the  department  of  chemistry. 
The  chemistry  of  the  urine,  therefore,  will  not  be  taken  up  here. 

This  chapter  is  limited  to  an  outline  of  a  few  experiments  deal- 
ing with  the  method  of  urine  secretion  and  excretion. 

1.  Movements  of  the  Ureter  and  Bladder. — Narcotize  a  rab- 
bit, lightly,  with  morphine.  Anaesthetize  with  ether,  just  suffi- 
ciently to  keep  the  animal  quiet. 

Prepare  absorbent-cotton  pads  soaked  in  hot  physiological  salt 
solution  for  protecting  the  abdominal  viscera  after  the  abdomen 
has  been  opened.  Open  the  abdomen  in  the  median  line.  Con- 
tinue the  incision  to  the  symphysis  pubis,  so  as  to  expose  the 
bladder. 

Note  the  form  of  this  organ  and  its  relation  to  the  surrounding 
viscera.  If  the  bladder  is  full,  stimulate  it  by  mechanical  irrita- 
tion or  by  the  application  of  a  tetanizing  induced  current.  Note 
the  character  of  its  contraction.  Does  it  continue  to  contract  and 
empty  itself  after  the  original  stimulus  has  ceased  to  act  ? 

Collect  the  urine  and  save  for  examination  and  comparison  in 
color,  clearness,  specific  gravity,  reaction,  and  constituents,  with  the 
normal  urine  of  man. 

Open  the  bladder  and  locate  the  entrances  of  the  two  ureters. 
Observe  these,  for  a  time,  for  the  passage  of  urine  into  the  bladder. 

Trace  the  left  ureter  to  the  kidney.  Dissect  this  out  from  its 
bed,  so  that  the  kidney,  ureter,  and  bladder  are  easily  observable. 
Observe  the  movements  of  the  ureter.  What  is  their  nature? 
How  do  they  compare  with  the  movements  of  the  intestines? 

[«S6] 


EXCRETION. 


What  is  the  normal  direction  of  the  ureter  movements  ?  Can  the 
movements  be  induced  in  response  to  a  mechanical  or  electrical 
stimulus  ?  Are  the  movements  rhythmical  or  irregular  ? 

2.  Urine  Flow.  Kidney  Volume. — Introduce  a  fine  glass  can- 
nula  into  the  ureter,  near  the  bladder  or  through  the  ureteral 
opening  into  the  bladder.  The 
speed  of  urine  flow  may  be  re- 
corded by  allowing  the  drops 
from  the  end  of  the  cannula  to 
fall  upon  a  lever,  made  for  the 
purpose  and  connected  with  a 
tambour  membrane.  This  tam- 
bour is  connected,  through  rubber 
tubing,  with  a  second  tambour 
whose  lever  is  arranged  to  write 
upon  the  smoked  paper  of  a 
slowly  revolving  drum. 

The  changes  in  volume  of  the 
kidney    may    be    determined    by 

means  of  a  plethysmograph  arrangement  known  as  an  oncometer. 
The  oncometer,  as  generally  used,  consists  of  a  metal  jacket  lined 
with  some  membrane  for  enclosing  the  kidney.  There  is  an  open- 
ing for  the  passage  in  and  out  of  the  kidney  vessels — artery,  vein, 
and  ureter.  The  space  between  the  membrane  and  the  jacket  is 
filled  with  oil.  This  space  is  connected  through  tubing  with  a 
piston  recorder  whose  lever  is  arranged  to  write  upon  the  smoked 
paper  of  a  revolving  drum.  In  this  way  a  curve  of  kidney  volume 
is  written.  A  simple  form  of  air  oncometer  is  shown  in  Fig.  39. 

The  kidney  is  partly  encased  in  a  rubber  balloon  inflated  with 
air.  The  changes  in  pressure  in  the  balloon  are  transmitted, 
through  rubber  tubing,  to  a  recording  tambour  or  bellows  re- 
corder. 

The  bellows  recorder  devised  by  Brodie  is  far  preferable  to  the 
tambour  as  a  recorder  of  volume  changes.  It  can  be  easily  made 
hi  the  laboratory  and  consists  of  two  rectangles,  hinged  with  thin 


FIG.  39.— Oncometer,  Simple  Form. 
B,  Metal  jacket ;  O,  opening  for  kid- 
ney vessels;  C,  rubber  balloon,  in- 
flated with  air,  partly  surrounding 
the  kidney  and  connected  through  T 
with  a  recording  tambour. 


LABORATORY  MANUAL  OF  PHYSIOLOGY. 

leather,  the  base  being  made  of  vulcanite  or  wood  and  perforated 
for  the  entrance  of  the  inlet  tube.  The  top  consists  of  a  light 
aluminum  frame  covered  with  paper.  The  sides  are  made  of 
peritoneal  membrane,  varnished  with  a  dilute  solution  of  boiled 
linseed  oil  to  make  them  airtight.1 

Place  the  kidney  in  the  oncometer,  cover  the  exposed  abdominal 
viscera  with  the  warm  cotton  pads  moistened  with  physiological 
salt  solution,  and  arrange  the  recording  apparatus  for  writing  on 
a  medium  slow  drum.  Arrange  the  recorder  for  urine  flow  under 
the  kidney-volume  recorder.  The  urine  should  also  be  collected 
for  examination,  later.  Be  careful  that  the  ureter,  the  renal  artery, 
and  renal  vein  are  not  obstructed  by  kinks.  Keep  the  ureter  from 
drying  by  moistening,  from  time  to  time,  with  physiological  salt 
solution. 

Observe  and  record  the  changes  in  kidney  volume  and  urine 
flow  for  a  period  of  twenty  minutes  or  one-half  hour.  Is  the  rate 
of  urine  flow  constant  during  this  time?  Are  there  any  changes 
in  the  volume  of  the  kidney?  How  do  urine  flow  and  kidney 
volume  correspond? 

3.  Blood  Pressure  and  Kidney  Volume. — Expose  the  carotid 
artery,  vagus  nerve,  depressor  nerve,  and  jugular  vein.  Introduce 
cannulae  into  the  artery  and  vein.  Pass  thread  loops  around  the 
nerves  for  convenience  in  handling.  Connect  the  artery  with  the 
mercury  manometer.  Record  blood  pressure  on  the  same  drum 
used  for  recording  kidney  volume  and  urine  flow.  Note  the  cor- 
respondence between  the  changes  in  blood  pressure  and  changes 
in  kidney  volume. 

(a)  Divide  one  vagus  nerve.     Stimulate  the  peripheral  end  with 
a  tetanizing  current  sufficiently  strong  to  cause  inhibition  of  the 
heart-beat.     Note  the  effect  upon  the  volume  of  the  kidney. 

(b)  Allow  the  blood  pressure  to  recover  from  the  effect   of   the 
vagus  stimulation.     Now  stimulate  the  depressor  nerve  with  a 
medium  strong  tetanizing  current  until  a  marked  depressor  effect 
is  obtained.     Note  the  effect  on  kidney  volume  and  urine  flow. 

1  For  further  details  see  Journal  of  Physiology,  vol.  xxvii.,  p.  473. 
[168]    ' 


EXCRETION. 

(c)  Allow  the  blood  pressure  to  return  to  normal.     Allow  the 
animal  to  inhale  a  few. whiffs  of   amyl  nitrite.     Note  the  effect 
upon  blood  pressure,  kidney  volume,  and  urine  flow. 

(d)  After  the  blood  pressure  has  again  returned  to  normal,  in- 
ject into  the  jugular  vein  one  cubic  centimetre  of  a  i  to   10,000 
solution  of  adrenalin  chlorid.     What  is  the  effect  upon  the  blood 
pressure,  kidney  volume,  and  urine  flow  ? 

(e)  After  the  blood  pressure  has  returned  to  normal,  connect 
the  vein  cannula  with  a  burette  containing  warm  physiological  salt 
solution.     Being  careful  that  there  are  no  air  bubbles  in  the  con- 
necting tubes,  allow  the  solution,  under  low  pressure,  to  run  slowly 
into  the  vein. 

Run  in  fifty  cubic  centimetres  of  the  solution.  Is  there  any 
noticeable  rise  of  blood  pressure  ?  Explain.  Is  there  any  change 
in  kidney  volume  or  urine  flow  ? 

(/)  Run  into  the  vein  fifty  cubic  centimetres  more  of  the  solu- 
tion. Note  any  effect  upon  blood  pressure,  kidney  volume,  or 
urine  flow. 

(g)  Repeat  the  perfusion,  using  fifteen  cubic  centimetres  of  a 
i-per-cent  urea  solution. 

4.  Intravenous  Injection  of  Dextrose. — Using  the  same  rab- 
bit as  in  the  previous  experiment,  or  a  fresh  animal  if  necessary, 
prepare   a    i -per- cent   dextrose  solution.      Warm   this  to  body 
temperature  and  slowly  inject  twenty  cubic  centimetres  of  this 
solution  into  a  vein.     Collect  the  urine  eliminated  before  and 
after  the  sugar  injection.     Test  both  with  Fehling's  solution  for 
reducing  substances.      Collect  samples    of  the  urine  every  ten 
minutes  after  the  beginning  of  the  sugar  injection.     When  does 
the  sugar  first  appear  in  the  urine  ?     When  does  it  cease  to  ap- 
pear in  the  urine  ? 

5.  Intravenous  Injection  of  Albumin. — Test  the  urine  for  al- 
bumin.    If  there  is  none  present,  inject,  into  a  vein,  ten  to  fifteen 
cubic  centimetres  of  a  i-per-cent  solution  of  egg  albumin  in  physio- 
logical salt  solution.     Examine  the  urine  for  albumin  at  intervals 
of  ten  minutes. 

[169] 


LABORATORY  MANUAL  OF  PHYSIOLOGY. 

6.  Intravenous  Injection  of  Peptone. — After   the  disappear- 
ance of  the  albumin  from  the  urine,  prepare  a  2-per-cent  peptone 
solution.     Inject  10  c.c.  of  this  solution  into  the  vein.     Collect 
the  urine  for  ten  minutes  or  until  enough  has  been  eliminated  for 
testing. 

Saturate  the  urine  with  ammonium  sulphate.  This  precipitates 
mucin,  albumin,  and  urates.  Filter  and  test  the  nitrate  for  pep- 
tones by  means  of  the  biuret  reaction. 

Do  peptones  occur,  normally,  in  the  blood  stream  ?  If  not,  what 
becomes  of  the  peptones  that  are  absorbed  from  the  gastric  and 
intestinal  mucous  membranes  ?  Is  peptone  ever  found  as  an  ab- 
normal constituent  of  the  urine  ?  Under  what  pathological  con- 
ditions may  peptonuria  occur? 

7.  Effect  of  Peptone  on  the  Coagulation  of  the  Blood. — Iso- 
late and  introduce  a  cannula  into  one  carotid  artery.     Open  the 
clamp  on  the  artery  and  collect  10  or  15  c.c.  of  blood  in  a  small 
test  tube.     Note  the  time  taken  for  solidification  of  the  shed 
blood. 

Now  inject  15  to  20  c.c.  of  the  peptone  solution  into  the  vein. 
In  three  or  four  minutes  open  the  artery  clamp  again ;  allow  2  or 
3  c.c.  to  escape,  and  then  collect  in  a  small  test  tube  10  to  15  c.c. 
of  blood.  Compare  the  coagulability  of  this  second  sample  with 
that  of  the  first  portion  of  blood  shed.  What  is  the  effect  of  pep- 
tone upon  the  coagulability  of  the  blood? 


CHAPTER  X. 

SENSATION. 

AN  organism  is  brought  into  relation  with  its  environment 
through  its  irritability  to  external  stimuli.  This  property,  of  irri- 
tability, is  common  to  all  protoplasm.  As  the  organism  increases 
in  complexity  from  single-celled  individuals  to  individuals  con- 
sisting of  groups  of  cells,  this  property  of  irritability  or  sensa- 
tion becomes  differentiated  into  a  variety  of  sensations,  depending 
upon  the  part  of  the  external  surface  or  special  end-organ  stimu- 
lated and  the  nature  of  the  stimulus. 

Conscious  sensation  first  occurs,  so  far  as  we  know,  in  those 
animals  provided  with  a  nervous  system  and  brain.  The  sensory 
impulse  is  conducted  over  nerve  pathways  to  the  sensory  portions 
of  the  cerebral  cortex,  and  there  interpreted  in  terms  of  sensation 
and  corresponding  judgments  formed. 

All  sensations  occur  as  a  result  of  some  form  of  stimulus  applied 
to  the  outer  body  envelope  and  its  connection  through  afferent 
nerves  with  the  centres  of  consciousness  in  the  brain.  For  the  re- 
ception and  transmission  of  certain  stimuli,  the  outer  envelope  has 
become  markedly  modified,  as,  for  example,  the  receiving  appara- 
tus for  audition  and  vision. 

The  localities  for  the  reception  of  certain  sensory  impressions 
are  limited  to  certain  sharply  defined  areas.  These  include  the 
end-organs  of  taste,  smell,  sight,  and  hearing.  Others  have  a  wide 
distribution  over  the  entire  cutaneous  surface  and,  to  a  lesser 
degree,  over  the  mucous  surfaces.  Such  are  the  tactile  sense,  the 
sense  of  temperature,  the  pain  sense,  and  the  pressure  sense.  The 
so-called  muscular  sense  also  has  a  wide  distribution. 

All  parts  of  the  body  are  brought  into  relation  with  the  central 
nervous  system  through  afferent  or  centripetal  nerves.  Only  part 


LABORATORY  MANUAL  OF  PHYSIOLOGY. 

of  these  sensations,  however,  are  commonly  brought  into  the  realm 
of  consciousness.  The  majority  of  such  impulses,  from  the  viscera, 
for  example,  are  either  lost,  through  diffusion  in  the  subsidiary 
parts  of  the  nervous  system,  or  are  transferred,  as  corresponding 
efferent  impulses,  to  complete  the  formation  of  reflex  arcs. 

When  such  impulses  become  abnormally  intense,  so  as  to  over- 
come the  resistance  in  the  longer  nerve  pathways  sufficiently  to 
reach  the  realm  of  cerebral  consciousness,  the  subjective  sensations 
are  either  vague  and  indefinable,  other  than  as  a  feeling  of  discom- 
fort or  pain  somewhere  in  the  region  involved,  or  they  are  referred, 
as  pain,  to  some  part  of  the  cutaneous  surface  whose  afferent  nerve 
distribution  corresponds  to  the  same  cord  segment  as  the  efferent 
nerve  distribution  of  the  visceral  area  involved.  Such  reference  of 
a  sensory  impression  occurs,  probably,  for  the  reason  that  the  sen- 
sorium  is  in  the  habit  of  receiving  impulses  from  the  skin  area  and 
not  from  the  visceral  area ;  and  where  the  same  terminal  neurons 
transmit  the  impressions  from  the  two  sources,  the  sensation  is 
referred  to  the  area  from  which  the  impulses  more  usually  come. 

The  nature  of  the  conscious  impression  depends,  not  so  much 
upon  the  character  of  the  stimulus  applied,  as  upon  the  peripheral 
area  stimulated,  the  afferent  nerve  involved,  and  the  brain  area  to 
which  the  impulse  goes.  Thus,  a  stimulation  of  the  optic  nerve, 
whether  it  be  mechanical,  electrical,  or  through  the  impact  of  light 
waves  upon  the  retina,  causes  a  sensation  of  light;  stimulation 
of  the  olfactory  nerves  gives  a  sensation  of  smell;  and  of  the  taste 
nerves,  of  taste. 

The  cutaneous  surface  itself  has  been  mapped  out  into  areas 
or  spots  which  are  irritable  to  stimuli  of  various  kinds.  Thus, 
there  are  spots  which  respond  to  stimuli  by  a  tactile  sensation, 
others  which  are  irritable  to  heat,  others  to  cold,  and  others  to 
stimuli  which  give  a  sensation  of  pain,  independent  of  temperature 
or  tactile  sensation. 

Quantitative  Relation  between  Stimulus  and  Sensation. — In  order 
that  a  stimulus  may  be  effective  in  producing  a  sensation,  its 
intensity  must  exceed  a  certain  minimum  value.  This  minimum 


SENSATION. 

is  sometimes  spoken  of  as  the  threshold  value  of  the  stimulus. 
This  threshold  value  is  a  variable  quantity,  varying  for  different 
individuals  and  for  the  same  individual  at  different  times.  It  de- 
pends partly  upon  the  condition  of  the  end-organ  and  the  over- 
lying integument,  and  partly  upon  the  receptivity  of  the  sensory 
cerebral  area  involved. 

If  the  intensity  of  the  stimulus  is  increased  progressively  above 
the  threshold  value,  the  intensity  of  the  sensation  increases  also, 
up  to  a  certain  maximum,  beyond  which  an  increase  in  the  strength 
of  the  stimulus  produces  no  further  increase  in  the  intensity  of 
the  sensation.  This  maximum  occurs  with  comparatively  weak 
stimuli.  The  range  of  sensory  variation  is,  therefore,  not  large. 
Between  the  maximum  and  minimum  a  variation  in  stimulus  is 
accompanied  by  a  variation  in  sensation.  This  variation  cannot 
be  measured  by  the  subject  of  the  sensation.  He  can  tell  that  one 
stimulus  is  stronger  or  weaker  than  another,  but  not  how  much 
stronger  or  how  much  weaker. 

An  increase  of  the  stimulus  above  the  maximum  of  sensory 
interpretation  very  rapidly  fatigues  the  sense  organ.  Even  with 
weak  stimuli,  the  sensory  apparatus  rapidly  tires. 

Weber's  Law. — E.  H.  Weber,  the  first  to  make  systematic  obser- 
vations along  these  lines  (1831),  formulated  the  following  conclu- 
sion, which  has  since  been  known  as  Weber's  law:  "An  increase  in 
a  stimulus  sufficient  to  call  forth  a  conscious  increase  in  the  sensa- 
tion must  always  bear  the  same  ratio  to  the  original  strength  of 
stimulus  to  which  it  is  added." 

For  example,  if  to  a  weight  of  i  it  is  necessary  to  add  a  weight 
J  in  order  that  the  subject  of  the  experiment  may  detect  a  differ- 
ence, then,  if  a  weight  of  10  is  used,  the  added  increment  necessary 
to  produce  an  increase  of  sensation  will  be  10  divided  by  3. 

I.  CUTANEOUS  SENSATION. 

1.  Tactile  Sense. — To  map  out  the  touch-spots  in  a  certain 
region  of  skin,  some  form  of  instrument,  known  as  an  aesthesiom- 
eter,  is  used.  A  simple  form  of  lesthesiometer  is  made  by  fasten- 


LABORATORY  MANUAL  OF  PHYSIOLOGY. 

ing  a  hair  at  right  angles  to  the  end  of  a  wooden  handle  by  means 
of  a  bit  of  sealing-wax.  If  this  hair  is  pressed,  perpendicularly, 
against  the  skin,  it  will  exert  a  certain  pressure,  depending  upon 
the  thickness  and  character  of  the  hair.  This  pressure  can  be 
determined,  for  any  hair,  by  pressing  it  against  one  scale  pan  of 
a  balance  and  finding  the  largest  weight  that  can  be  lifted  in  this 
way. 

(a)  Prepare  a  number  of  hair  aesthesiometers,  using  hairs  of 
different  lengths  and  thicknesses  and   estimating   their   pressure 
values. 

(b)  Gently  touch  the  end  of  a  hair  on  the  back  of  the  hand. 
Note  the  sensitiveness  of  the  hair  as  a  touch  organ.     The  end 
organs  of  touch  are  arranged  in  radiating  lines  about  the  roots  of 
the  hairs.     The  hairs  act  as  levers,  the  long  arm  projecting  above 
the  skin  surface  and  the  short  arm  making  pressure  against  the 
nerve  endings.     In  this  connection   consider  the  so-called  touch 
hairs  of  the  cat  and  other  animals. 

(c)  Shave  the  skin  of  the  back  of  the  hand,  and,  starting  at  the 
hair  follicle,  map  out  the  touch-spots  in  an  area  of  2  sq.  cm.     Start 
with  a  test  hair  of  least  pressure  and  increase  the  pressure  until 
sensation  is  produced.     The  subject  of  the  experiment  should  be 
blindfolded  and  instructed  to  say  yes  immediately  upon  feeling  the 
application  of  the  test  object. 

Record  the  threshold  value  of  the  stimulus  needed  to  produce 
sensation  in  this  region.  Record,  also,  the  number  of  touch-spots 
present  in  the  area  of  skin  tested. 

What  is  the  arrangement  of  the  touch-spots  in  relation  to  the 
hair  follicle? 

(d)  Shave  the  skin  of  the  back  of  the  leg  and  map  out  the  touch- 
spots  and  determine  the  threshold  value  of  the  stimulus,  in  the 
same  way  as  was  done  for  the  skin  of  the  back  of  the  hand. 

(e)  Repeat  the  experiment  for  the  skin  of  the  abdomen,  near 
the  median  line  and  some  distance  from  the  median  line. 

(/)  Test  the  touch  sensation  of  the  skin  of  the  back,  over  the 
shoulder. 

[174] 


SENSATION. 

(g)  Map  out  the  touch-spots  of  the  cheek,  starting  near  the  lobe 
of  the  ear  and  proceeding  to  the  angle  of  the  mouth.  In  what  part 
of  this  area  are  the  touch-spots  most  numerous  ? 

(k)  Test  the  mucous  membrane  of  the  upper  and  lower  lips  in 
the  same  way. 

(i)  Test  the  palmar  surfaces  of  the  finger  tips;  of  the  hand. 

How  do  the  threshold  values  of  the  efficient  stimuli,  for  the 
various  regions  tested,  compare?  Is  there  any  relation  between 
the  number  of  touch-spots  and  the  mobility  of  the  regions  tested  ? 

(j)  Repeat  experiments  (a)  to  (i),  using,  instead  of  the  test 
hairs,  a  series  of  weights  of  the  same  surface  area  (about  4  sq.  mm.) . 
Begin  with  a  weight  of  0.0005  gm.  and  increase  until  the  sensation 
is  obtained.  How  do  the  threshold  values  compare  with  those  ob- 
tained with  the  test  hair  ?  In  the  first  series  of  experiments,  single 
touch-spots  were  stimulated.  In  the  second  series,  a  number  of 
touch-spots  were  stimulated  simultaneously,  the  number  varying 
with  the  region  of  skin  to  which  the  stimulus  was  applied. 

The  efficacy  of  any  particular  stimulus  will  depend,  to  a  large 
extent,  upon  the  number  of  touch-spots  in  the  area  stimulated, 
the  proximity  of  the  end  organs  to  the  skin  surface,  and  the  deforma- 
tion of  the  skin  effected  by  the  stimulus.  The  temperature  of  the 
weights  employed  should  be  approximately  that  of  the  skin. 

(k)  Take  a  number  of  pieces  of  flat  cork,  cut  into  strips  about 
3  cm.  long,  i  cm.  wide,  and  i  cm.  thick,  and  pass  blunted  needles 
through  the  ends  of  the  strips  so  that  the  distance  between  the 
needle-points  of  the  different  pairs  varies  from  i  to  25  mm. 

With  a  blindfolded  subject,  test  the  ability  of  the  skin  of  various 
regions  to  detect  the  application  of  the  different  pairs  of  needles  as 
two  separate  stimuli.  The  subject  should  answer,  immediately 
upon  the  application  of  the  stimulus,  one  or  two,  as  the  sensation 
is  that  of  one  point  or  two  points. 

In  this  way  test  the  sensitiveness  of  the  skin  of  the  finger-tips, 
the  back  of  the  hand,  the  shoulder-blade,  the  forehead,  the  cheek, 
the  lips,  the  tip  of  the  tongue,  the  skin  of  the  thigh  in  its  long  axis, 
the  skin  of  the  thigh  in  its  short  axis. 


LABORATORY  MANUAL  OF  PHYSIOLOGY. 

In  which  of  these  areas  are  the  two  points  of  the  test  needles 
distinguished  as  separate,  when  brought  nearest  together  ?  Why  ? 

In  which  axis  of  a  limb  are  the  two  points  of  the  test  object  most 
readily  distinguished?  Explain. 

Is  there  any  after-sensation  in  any  of  the  skin  areas  stimulated  ? 

(i)  Stimulation  of  the  touch-spots,  through  pressure,  occurs  as 
a  consequence  of  deformation  of  the  skin.  If  a  constant  pressure 
is  applied  to  all  parts  of  a  skin  surface  equally,  there  is  no  sensa- 
tion of  touch.  This  may  be  accomplished  by  immersing  the  hand 
in  a  vessel  of  water  at  the  same  temperature  as  that  of  the  skin. 
So  long  as  the  hand  remains  quiet  and  the  water  is  still,  there  is  no 
sensation  of  touch,  except  at  the  junction  of  air  and  water  at  the 
surface  of  the  liquid. 

2.  Temperature  Sense. — Temperature  sensation  is  of  two 
kinds,  sensation  of  cold  and  sensation  of  heat.  There  are,  ap- 
parently, separate  sets  of  nerve  fibres  and  endings  for  these  two 
sensations,  since  certain  skin  areas  are  irritable  to  cold  objects  but 
not  to  warm,  and  others  are  irritable  to  objects  warmer  than  the 
skin  but  not  to  cold  stimuli. 

(a)  These  areas  may  be  mapped  out  as  so-called  warm  and  cold 
spots  by  a  method  similar  to  that  used  in  mapping  out  the  touch- 
spots.     Take  a  metal  cannula,  drawn  to  a  fine  point — a  small  ar- 
tery cannula  may  be  used — and  run  hot  water  through  it  for  a  time. 
Choose  an  area  of  skin,  about  4  sq.  cm.  on  the  back  of  the  hand, 
for  testing.     Bring  the  tip  of  the  cannula  sufficiently  near  the  skin 
to  obtain  the  sensation  of  heat  without  mixing  this  with  the  touch 
sense.     Mark  those  points  where  the  heat  sensation  occurs,  with 
a  fine-pointed  colored  pencil. 

(b)  Go  over  the  same  area  of  skin  as  in  (#),  running  cold  water 
through  the  cannula  instead  of  hot  as  before.     Mark  the  cold  spots 
with  a  fine  pencil  of  another  color  than  that  employed  in  marking 
the  warm  spots. 

(c)  A  rough  estimate  of  the  temperature  sense,  in  various  skin 
areas  of  the  body,  may  be  made  by  filling  two  test  tubes  of  small 
calibre  with  hot  and  cold  water,  respectively.     These  are  applied, 


SENSATION. 

alternately,  to  the  same  skin  areas,  and  the  sensation  experienced 
is  recorded  and  compared  with  that  obtained  in  other  skin  areas. 
(d)  With  a  pair  of  blunt-pointed  dividers,  determine  the  nearest 
distance  between  the  points  at  which  they  are  detected  as  two. 
Now  warm  the  points  and  repeat  the  experiment.  Is  the  distance 
increased  or  decreased  at  which  the  points  are  separately  felt  by 
the  skin? 


12  [177] 


CHAPTER  XL 

VISION. 
I.  DISSECTION  OF  THE  EYE. 

1.  Appendages. — (a)  Examine  the  specimen  before  you,  tracing 
out  the  ocular  and  palpebral  conjunctiva,  noting  the  plica  semi- 
lunaris  and  the  caruncula.     How  do  the  latter  compare  in  relative 
size  with  the  human  structures  ?     Locate  and  describe  the  puncta 
lachrymalia  and  the  openings  of  the  lachrymal  ducts.     How  many 
are  there?     Is  your  specimen  a  right  or  left  eye? 

(b)  Observe  carefully  the  appendages,  locating  the  tarsal  carti- 
lages, Meibomian,  sebaceous,  and  lachrymal  glands.  Observe  the 
recti  and  oblique  muscles  and  their  actions  on  the  eyeball.  Ob- 
serve the  entrance  of  the  optic  nerve. 

2.  By  pinning  down  the  flaps  of  the  conjunctiva,  fix  the  eyeball 
to  the  board,  the  cornea  downward.     Then  dissect  out  the  four 
recti  and  two  oblique  muscles,  observing  the  capsule  of  Tenon. 

Without  injuring  important  vessels  and  nerves,  remove  the 
heavy  retractor  muscle.  Locate  and  describe  the  vence  vorticosce. 
How  many  are  there?  Find  the  anterior  ciliary  arteries.  How 
many  are  there  ?  What  structures  do  they  supply  ?  Find  the  two 
long  ciliary  arteries,  the  short  posterior  ciliary  arteries,  and  the 
ciliary  nerves. 

3.  Eyeball. — (a)  Fix  the  eyeball  to  the  board,  cornea  up,  pin- 
ning down  the  dissected  muscles  as  guys.     After  having  observed 
the  cornea  remove  it  with  heavy  scissors,  near  the  corneo-scleral 
margin. 

(fy  Through  the  opening  thus  made,  examine  the  iris.  Where 
is  the  posterior  chamber  ? 

(r)  Holding  the  margin  of  the  cornea  with  strong  forceps,  dissect 


VISION. 

the  sclerotic  coat  free  from  the  choroid,  for  about  3  mm.  posterior 
to  the  angle  of  the  anterior  chamber.  Between  the  insertions  of 
the  recti  muscles,  locate  four  points  on  the  margin  from  which 
incisions  may  be  made  antero-posteriorly.  From  these  points, 
make  the  incision  posteriorly  as  far  as  the  equator  of  the  eyeball. 
Dissect  each  flap  free  from  the  underlying  choroid.  After  having 
removed  the  pins  fixing  the  recti  muscles,  draw  the  flaps  back  and 
fix.  Observe  the  iridal  and  ciliary  portions  of  the  choroid. 

(d)  With  a  fine  forceps,  grasp  the  margin  of  the  iris  and  with 
small  scissors  cut  out  a  sector  with  the  ciliary  body  as  a  base. 
Study  the  posterior  chamber,  suspensory  ligament,  and  the  anterior 
surface  of  the  ciliary  body. 

(e)  Make  a  circular  incision  with  small  scissors,  severing  choroid 
and  retina  at  about  the  line  of  the  ora  serrata.     Lift  off  from  the 
vitreous   humor  the  whole  ciliary  apparatus,  placing  it  upon  a 
plate,  anterior  surface  downward.     Observe  the  posterior  aspect 
of  the  ciliary  body.     Describe  the  lens  carefully,  making  a  cross- 
section.     Can  you  discern  its  capsule? 

(I)  Observe  the  retina,  as  seen  through  the  vitreous,  locating 
the  entrance  of  the  optic  nerve.  Can  you  locate  the  f  ovea  centralis  ? 

II.  PHYSIOLOGICAL  OPTICS. 

Light  is  propagated  from  a  luminous  point  in  every  plane  and 
in  every  direction,  in  straight  lines.  These  lines  of  direction  are 
called  rays.  Rays  travel  with  the  same  rapidity  so  long  as  they 
remain  in  the  same  medium;  the  denser  the  medium  the  slower  the 
passage  of  light  through  it.  The  divergence  of  the  rays  of  light  is 
proportionate  to  the  distance  from  which  they  come. 

Rays  of  light  proceeding  from  infinity  are  parallel.  In  dealing 
with  rays  of  light  which  enter  the  eye,  it  will  be  sufficiently  accurate 
to  consider  them  parallel  when  they  proceed  from  a  point  more 
than  six  metres  distant. 

A  ray  of  light,  meeting  with  a  body,  may  be  absorbed,  reflected, 
or,  if  the  body  is  transparent,  refracted.  In  dealing  with  the  eye, 
it  is  necessary  to  consider  only  the  latter. 


LABORATORY  MANUAL  OF  PHYSIOLOGY. 

1.  Refraction. — A  ray  of  light  passing  through  one  transparent 
medium  into  another  of  different  density  is  bent  or  refracted, 
unless  the  ray  falls  perpendicular  to  the  surface  of  the  denser 
medium.  The  ray  is  spoken  of  as  incident  before  entering  the 
second  medium,  and  emergent  after  leaving  it. 

Upon  entering  a  denser  medium,  the  ray  is  refracted  toward  the 
perpendicular  and  from  the  perpendicular  upon  entering  a  rarer 
one.  Reflection  accompanies  refraction,  the  ray  dividing  at  the 
point  of  incidence. 

THE  INDEX  OF  REFRACTION  is  the  relative  resistance  of  a  sub- 
stance to  the  passage  of  light.  Air  is  taken  as  a  standard  and  is 
called  i.  The  index  of  refraction  of  water  is  1.3,  of  glass,  1.5. 
The  diamond  has  the  highest  refractive  index,  which  is  2.4. 

LENSES. — A  lens  is  a  transparent  substance,  usually  glass,  bound- 
ed by  two  curved  surfaces,  or  by  one  plane  and  one  curved  surface. 


$ 

FIG.  40.— «,  Object ;  /',  image ;  n,  nodal  point ;  L,  lens ;  F,  F/,  and/,/,  conjugate  foci. 

It  may  be  regarded  as  a  series  of  prisms.  In  a  convex  lens  the 
bases  are  directed  toward  the  centre,  and  in  a  concave  lens  the 
bases  are  directed  away  from  the  centre.  Rays  of  light  passing 
through  a  convex  lens  are  made  to  converge.  Those  passing 
through  a  concave  lens  are  made  to  diverge. 

The  point  to  which  rays  converge,  after  passing  through  a  con- 
vex lens,  is  its  focus.  The  principal  focus  of  a  convex  lens  is  its 
focus  for  parallel  rays. 

When  rays  of  light  diverge  from  any  point  nearer  than  infinity, 
they  are  brought  to  a  focus  at  a  point  beyond  the  principal  focus. 

[180] 


VISION. 

The  point  from  which  they  diverge  and  the  point  to  which  they 
converge  are  called  conjugate  foci.  As  one  approaches  the  lens, 
the  other  recedes,  and  vice  versa. 

By  means  of  a  candle,  determine  the  principal  focus  of  the  con- 
vex lens  before  you. 

The  foci  of  concave  lenses  for  parallel  or  divergent  rays  are 


FIG.  41.- Described  in  text. 

virtual  or  negative.  They  are  the  points  from  which  rays  seem  to 
diverge  after  passing  through  the  lens  (see  Fig.  41,  F). 

Parallel  rays  of  light,  falling  upon  a  concave  lens,  are  diverged. 
If  these  rays  were  traced  backward,  they  would  seem  to  diverge 
from  a  point  nearer  the  lens  (see  Fig.  41,  F). 

The  conjugate  foci  of  concave  lenses  are  also  virtual  and  found 
in  a  similar  manner.  Find  them. 

Formation  of  images.  The  image  of  an  object  is  the  collection 
of  the  foci  of  its  principal  points. 


FIG.  42.— Described  in  text. 

SIMPLE  DIOPTRIC  SYSTEM. — The  simplest  form  of  a  dioptric 
apparatus  consists  of  two  media  of  different  refractive  indices, 

[181] 


LABORATORY  MANUAL  OF  PHYSIOLOGY. 

separated  by  a  spherical  surface.  In  such  an  apparatus,  the 
optical  properties  depend  upon  the  curvature  of  the  surface  and 
the  refractive  power  of  the  media.  Such  an  apparatus  is  shown 
in  the  accompanying  figure  (Fig.  42).  The  line  pr  represents  a 
curved  surface  separating  media  of  different  refractive  power,  the 
lens  being  on  the  left  The  line  oa,  falling  perpendicularly  upon  the 
surface  at  3,  passes  through  the  centre  of  the  sphere,  6.  The  line 
o-a  is  the  optical  axis.  All  the  lines  that  cut  the  surface  normally, 
such  as  o-dj  c-y,  and  u-i,  undergo  no  refraction  and,  continuing  in 
straight  lines,  cross  at  6,  which  is  the  nodal  point.  All  of  the  rays 
are  refracted.  All  rays,  parallel  to  the  optical  axis,  passing  through 
the  lens  will  be  bent  so  as  to  meet  at  m,  which  is  the  posterior 
principal  focus.  The  anterior  principal  focus  is  at  b,  in  the  first 
medium  and  in  front  of  the  lens.  Rays  of  light,  such  as  b-i-t, 
passing  from  it,  are  so  refracted  that  they  become  parallel  to  the 
optic  axis.  The  principal  point  is  the  point  where  the  optic  axis 
cuts  the  surface.  The  posterior,  anterior,  nodal,  and  principal 
points  are  the  cardinal  points  of  an  optical  system. 

THE  EYE  AS  AN  OPTICAL  INSTRUMENT. — Having  reviewed  the 
general  optical  principles  concerning  the  refraction  of  light  and 
the  formation  of  images  by  convex  lenses,  we  now  come  to  the  eye 
as  an  optical  instrument.  Rays  of  light,  as  they  enter  the  eye 
encounter  not  one  refracting  medium  as  in  the  simple  dioptric 
system,  but  five,  namely: 

Tears, 

Cornea, 

Aqueous  humor, 

Lens, 

Vitreous  humor. 

The  indices  of  refraction  of  these  various  media  are  such  that 
parallel  rays  of  light,  entering  a  normal  eye,  are  brought  to  a  focus 
upon  the  retina.  For  the  sake  of  simplicity,  they  may  be  looked 
upon  as  equal  to  a  convex  lens  of  about  twenty-three  millimetres 
focus.  However,  a  ray  of  light  falling  upon  the  cornea  does  not 
follow  the  same  simple  direction  it  would,  were  it  to  pass  through  a 


VISION. 

single  medium.  Instead,  the  eye  must  be  regarded  as  a  compound 
refracting  system,  composed  of  a  spherical  surface  and  a  biconvex 
lens. 

The  cardinal  points  are 
Two. principal  points, 
Two  nodal  points, 
Two  principal  foci. 

In   the  diagram,  Fig.  43,  the  cardinal   points   are   shown   all 
upon  the  optic  axis,  f-a.     At  6,  two  principal  points,  situated  so 


FIG.  43.— Described  in  text. 

close  together  in  the  anterior  chamber  that  they  may  be  regarded 
as  one.  At  /  is  the  first  principal  focus,  and  at  a  the  second. 
The  nodal  points  correspond  nearly  to  the  optical  centre  of  the 
refractive  system.  Rays  passing  through  these  points  are  not 
refracted.  They  are  situated  about  7  mm.  behind  the  cornea. 

THE  FORMATION  OF  RETINAL  IMAGES.  A  luminous  point 
placed  above  the  principal  axis  has  its  image  formed  upon  the 
retina  below  this  axis,  and  vice  versa.  Replace  these  points  by  an 
object  and  the  same  thing  occurs.  The  retinal  image  is,  as  it 
were,  a  mosaic,  composed  of  innumerable  foci  of  the  object. 

Construct  a  simple  diagram  of  the  human  eye,  showing  the 
formation  of  an  image,  say  an  arrow  or  a  candle,  upon  the  retina. 
Is  the  image  erect  or  inverted  ?  If  inverted,  why  do  we  see  it  erect  ? 

The  human  eye  has  aptly  been  compared  to  a  camera,  the 
refracting  media  representing  the  camera  lenses,  and  the  retina 
its  sensitive  plate. 

[183] 


LABORATORY  MANUAL  OF  PHYSIOLOGY. 

ADAPTATION  OF  THE  EYE  FOR  DISTANCE. 

In  the  camera,  however,  it  is  necessary  to  adjust  the  instrument 
by  backward  and  forward  movements  of  the  lenses.  In  the  eye, 
this  adjustment  is  brought  about  by  changes  in  convexity  of  the 
lens,  or  accommodation.  Accommodation  is,  therefore,  the  func- 
tional adaptation  of  the  eye  to  distance. 

If  the  entire  optical  apparatus  of  the  eye  were  rigid  and  fixed, 
how  could  objects  at  various  distances  be  seen  clearly  ?  Explain 
what  takes  place  when  the  eye  accommodates. 

Take  'a  sharp-pointed  pencil  in  each  hand.  With  one  eye 
closed,  hold  the  points  in  a  direct  line  of  vision  before  the  other 
eye — one,  about  twenty  centimetres  distant,  and  the  other  a  full 
arm's  length.  Focus  on  the  nearer  pencil.  Is  the  image  of  the 
distant  one  clear  ?  Focus  upon  the  farther  pencil.  Is  the  image 
of  the  nearer  one  clear? 

The  near  point,  or  punctum  proximum,  is  the  nearest  point  to 
the  eye  to  which  objects  may  be  brought  and  still  be  seen  clearly. 
It  averages  about  1 2  cm.  At  this  point  the  accommodation  is  most 
active. 

Determine  your  own  near  point. 

The  far  point,  or  punctum  remotum,  is  the  farthest  point  at 
which  objects  may  be  seen  clearly  by  the  normal  eye. 

The  range  of  accommodation  is  the  difference  between  the  punc- 
tum proximum  and  the  punctum  remorum. 

Determine  your  own  range  of  accommodation. 

ADAPTATION  OF  THE  EYE  FOR  DIRECTION. 

As  the  eye  can  functionally  adjust  itself  to  distance,  it  can  also 
change  the  direction  of  its  visual  axis  from  one  object  to  another, 
or  can  follow  objects  moving  within  its  field  of  vision. 

Two  students  may  work  together,  one  as  observer  and  the  other 
as  subject. 

(a)  MONOCULAR  FIXATION. — The  observer  and  subject  being 
seated  opposite  each  other,  let  the  subject  close  or  screen  one  eye. 


VISION. 


(i)  Hold  any  small  object  directly  in  front  of  the  subject  and  have 
him  fix  his  eye  upon  it  constantly.  Move  the  object  quickly 
toward  the  subject's  left  and  notice  the  immediate  fixation  of  the 
object  in  its  new  position.  What  muscles  are  brought  into  action, 
in  this  movement?  (2)  Move  the  object  quickly  to  the  right, 
upward,  downward,  and  diagonally,  noticing  the  immediate  fixa- 
tion in  all  the  fields.  What  muscles  are  brought  into  action  in 
each  position,  and  are  all  movements  equally  rapid  ?  (3)  Bring 
the  object  exactly  in  front  about  one  metre  distance  and  note  the 
range  of  lateral  movement  without  causing  any  appreciable  change 
in  the  visual  axis.  (4)  Bring  the  object  to  the  central  position  and 
move  it  very  slowly  outward  in  various  directions  and  observe 
whether  the  changes  of  d, 

direction  of  the  visual 
axes  are  equally  slow  and 
regular. 

(b)  BINOCULAR  FIXA- 
TION.— Convergence.  —  It 
was  probably  noticed  dur- 
ing the  above  exercises 
that,  though  one  eye  was 
screened,  it  shared  in  all 
movements  with  its  fel- 
low. With  both  eyes 
open,  let  the  subject  fix 
a  small  object,  held  about 
one  metre  distant.  Let 
the  observer  move  the 
object  slowly  in  all 
fields,  downward,  upward,  laterally,  and  around,  observing  the 
perfect  continuous  fixation  with  both  eyes. 

What  muscles  or  pairs  of  muscles  are  involved  in  the  movements 
in  the  different  directions  ?  If  any  variations  are  noticed  in  the 
subjects  examined,  describe  them. 

STEREOSCOPIC  VISION. — Binocular  Single  Vision. — By  this  is 


FlG.  44.-  a,  6,  Two  objects,  the  images  of  which 
(a,,  bl  and  a3,  *,)  fall  on  corresponding  parts  of 
both  retinae,  K  and  /?,. 


LABORATORY  MANUAL  OF  PHYSIOLOGY. 

meant  the  union,  in  one  single  impression,  of  images  received 
simultaneously  on  both  retinae.  The  external  ocular  muscles 
maintain  the  visual  axes  parallel,  so  that  impressions  of  an  object 
fall  on  correspondingly  identical  points  of  both  retinae  (see  Fig. 

44)- 

What  happens  when  paralysis  of  one  ocular  muscle  destroys 

balance  ?     Show  by  diagram. 

CONVERGENCE. — Let  the  subject  fix  his  vision  upon  an  object 
within  an  arm's  length,  and  then  upon  some  object  more  than  six 
metres  distant  but  in  the  same  line  of  vision. 

What  change  takes  place  in  relation  of  the  visual  axes  to  each 
other? 

Hold  an  object  one  metre  distant,  directly  in  front  of  the  subject. 
Move  it  directly  toward  the  subject's  eyes.  Note  the  convergence 
of  the  visual  axes. 

What  change  takes  place  in  the  pupil?  What  muscles  are  in- 
volved in  the  act?  Recall  the  innervation  of  the  pupil  and  the 
internal  recti. 

OPTICAL  DEFECTS. 

The  eye  is  not  a  perfect  optical  instrument,  since  it  is  not  exactly 
centred  and  possesses  in  small  degree  chromatic  and  spherical 
aberration. 

By  chromatic  aberration  is  meant  that  different  rays  of  the 
spectrum  are  bent  to  different  degrees.  For  instance,  violet  rays, 
which  are  more  refrangible  than  red,  have  their  focus  near  to  the 
lens.  In  the  manufacture  of  optical  instruments,  this  is  overcome 
by  combining  a  convex  with  a  plano-concave  lens.  Practically  the 
same  arrangement  exists  in  the  eye,  and  this,  combined  with  the 
rapid  accommodative  ability  of  the  lens,  makes  chromatic  aberra- 
tion a  negligible  quantity. 

By  spherical  aberration  is  meant  that  rays  of  light  which  traverse 
the  periphery  of  a  lens  are  brought  to  a  focus  sooner  than  those 
which  pass  nearer  the  centre.  The  iris  corrects  this  defect  by 
acting  as  a  diaphragm,  shutting  off  the  peripheral  rays. 

[186] 


VISION. 

MISCELLANEOUS  EXPERIMENTS. 

Blind  Spot. — On  a  white  card  make  a  small  black  cross,  and 
about  8  cm.  away,  in  a  horizontal  direction,  draw  a  black  dot. 
Looking  intently  at  the  dot,  one  eye  having  been  screened,  hold  the 
card  about  25  cm.  from  the  eye  and  then  move  it  slowly  toward  the 
eye.  At  what  distance  does  the  cross  disappear?  Why? 

Imperfect  Visual  Judgments. — Draw  one  line  (horizontal),  6  cm. 
long,  and  one  perpendicular  to  the  same  length  but  not  joining. 
Which  line  appears  the  longer  and  why  ? 

Make  three  black  dots  on  the  same  imaginary  horizontal  line 
and  equidistant  from  each  other.  Mark  them  x,  y,  and  z.  Con- 
nect x  and  y  with  a  series  of  equidistant  dots  of  the  same  size. 
Which  appear  farther  apart,  x  and  y  or  y  and  z  ? 

Draw  two  horizontal  lines  5  cm.  long.  On  the  upper  one  make 
arrow-heads  pointing  toward  each  other.  On  the  lower  make 
arrow-heads  pointing  away  from  each  other.  Which  line  appears 
the  longer? 

Draw  three  parallel  lines.  On  the  upper  one  draw  a  series  of 
short,  parallel  intersecting  lines,  cutting  the  longer  line  at  an  angle. 
Do  the  same  with  the  middle  line,  except  to  make  the  angle  of 
intersection  equal  and  opposite  to  that  of  the  first  series.  Prepare 
the  lower  line  the  same  as  the  upper  line.  Do  the  lines  still  appear 
parallel  ? 

Sanson-Purkinje  Images. — Darken  the  room.  Hold  a  lighted 
candle  a  little  to  one  side  and  in  front  of  the  subject's  eye.  The 
observer,  looking  at  the  eye  from  the  other  side,  sees  three  images 
of  the  flame.  The  first  and  brightest  is  a  small,  erect  image 
formed  by  the  anterior  convex  surface  of  the  cornea.  The  sec- 
ond, larger  and  less  distinct,  is  formed  by  the  anterior  convex  sur- 
face of  the  lens.  The  third,  smaller,  inverted,  and  indistinct,  is 
formed  by  the  posterior  surface  of  the  lens.  Let  the  subject  ac- 
commodate for  a  near  object.  Describe  the  change  in  relation 
that  takes  place  in  the  size  and  clearness  of  the  second  image  and 
its  proximity  to  the  first. 


LABORATORY  MANUAL  OF  PHYSIOLOGY. 

Duration  of  Impressions. — On  a  circular  white  disc,  mid- 
way between  the  periphery  and  centre,  fix  a  small  black  ob- 
long disc.  Rotate  it  rapidly.  Note  that  a  ring  of  gray  appears 
on  the  black,  showing  that  retinal  impressions  are  of  a  certain 
duration. 

Inversion  of  Shadows. — Make  three  pinholes  in  a  card,  close 
together  and  arranged  in  a  small  triangle.  Hold  the  card  about 
12  to  15  cm.  from  the  right  eye.  Look  through  the  holes  at  a 
bright  light.  Close  the  left  eye  and  hold  a  pin  in  front  of  the  right 
eye,  so  that  it  just  touches  the  lashes.  Note  that  an  inverted 
image  of  the  pin  will  be  seen  in  each  hole.  Retinal  images  are 
inverted.  Shadows  are  erect.  Therefore  the  latter,  upon  being 
projected  outward  into  space,  are  seen  inverted. 

NORMAL  VISION. 

Examination  of  Distant  Vision. — The  sense  of  sight  consists  of 
(i)  form  sense  (acuity),  (2)  light  sense,  (3)  color  sense. 

The  acuteness  of  direct  vision  is  measured  by  means  of  letters, 
sized  to  certain  definite  standards.  Those  devised  by  Snellen  are 
in  most  common  use.  Snellen  determined  the  normal  acuteness 
of  vision  to  be  the  power  of  distinguishing  letters  subtending  the 
visual  angle  of  5'.  The  letters  are  formed  of  strokes  whose  width 
is  one-fifth  the  size  of  each  letter,  hence  they  are 'seen  under  an 
angle  of  i'.  The  openings  in  the  letters,  and  the  spaces  between 
the  contiguous  strokes,  are  made  to  conform,  as  nearly  as  possible, 
to  the  same  angle. 

The  relation  of  the  size  of  the  letter  to  the  distance  at  which  it 
should  be  discerned  by  the  normal  eye  is  expressed  by  twice  the 
tangent  of  half  the  angle  of  5',  or,  0.001425.  The  size  of  the  letter, 
the  perception  of  which  constitutes  normal  vision  at  a  given  dis- 
tance, may  be  obtained  by  multiplying  the  distance  by  0.001425. 
On  this,  the  standard  letters  of  measuring  visual  acuity  have  been 
built  up. 

Practical  experience,  however,  has  shown  that  letters  constructed 
under  the  angle  of  5'  do  not  always  give  the  best  visual  acuity  of 

[188] 


VISION. 

which  the  subject  is  capable;  so  that  figures  constructed  on  the 
4'  basis  are  gradually  coming  into  use. 

For  recording  visual  acuity,  the  formula  V  =  —  is  used,  V 

standing  for  vision;  d,  for  the  distance  of  the  subject  from  the  test 
type;  D,  the  distance  from  which  it  should  be  read. 

In  practice,  the  acuteness  of  vision  is  found  by  determining  the 
smallest  type  the  subject  can  read  at  six  metres.  Normal  vision 
is  represented  by  the  symbol  f .  That  is  to  say,  that  at  six  metres 
the  subject  reads  the  test  line  marked  6.  If,  at  this  distance,  the 
subject  can  only  read  the  line  marked  12,  his  visual  record  would 
be  T6^,  and  so  on. 

In  instances  where  the  vision  of  the  subject  is  lowered  to  the 
extent  that  he  cannot  see  even  the  largest  test  types,  vision  is 
tested  by  the  ability  to  count  fingers  at  varying  distances.  For 
example,  V  —  fingers,  3  m.  If  vision  is  still  lower,  V  =  shadows 
or  light. 

Examination  of  Near  Vision. — This  includes  the  ability  of  the 
subject  to  read  print.  That  is  the  condition  of  accommodation. 
The  test  types  are  those  of  von  Jaeger  and  Snellen. 

Exercise. — Test  the  visual  acuity  of  each  other,  recording  your 
findings,  both  for  distant  and  near. 

Light  sense  is  the  power  possessed  by  the  retina  of  appreciating 
variations  in  the  intensity  of  light.  It  is  measured  by  the  photom- 
eter, which  consists,  essentially,  of  an  apparatus  by  which  the  in- 
tensity of  two  sources  of  light  may  be  compared. 

Color  Sense. — This  is  the  power  the  retina  has  of  distinguishing 
or  perceiving  colors,  or  the  impression  resulting  from  the  impact 
of  light  rays  having  different  refrangibilities. 

Holmgren's  test  consists  of  testing  the  power  of  a  subject  to 
match  various  colored  yarns.  Three  large  test  skeins,  namely,  (i) 
light,  pure  green,  (2)  rose-purple,  (3)  red,  are  given  the  patient, 
and  also  smaller  skeins,  comprising  various  shades  and  tints  of 
each  color.  He  is  requested  to  pick  out  the  colors  similar  to  his 
original  three  skeins.  If,  for  example,  he  is  red-blind,  he  will  not 


LABORATORY  MANUAL  OF  PHYSIOLOGY. 

*  red  m  the  purple  or  related  colors,  but  will  dassify  these 
te  reds  wul  be  confused  with  the  greens. 


the  ere,  in  a  state  of  rest, 

„*• »-  •» ^  ,   , , »•    .,,_,,__  +» 

-     -    _       .     .    • ..      •     :      .  - ; 

t   condition  of 


(tar  si-htedness)  is  that  form  of 
eye,  when  at  rest,  focusses  parallel  rays  of 
It  is  corrected  bj  placing  before  the  eye 
.    Why?    Make  a  diagram. 

arH9gfatedness)  is  that  fonn  of  ametropia  in  which 
at  T1p^tJ,  fc*  imy^  pnolifi  ravs  of  i^ht  in  front  of  the 
It  is  Maintrtl  bj  pbring  before  flic  eye  a  concave  fens. 
Why?    Make  a  dbgram. 

of  ametropia  in  which  the  eye,  when 
rajs  of  fight  upon  any  one  spot, 
rays  of  fight,  coming  through   different 

to  2  mOOQS  ffl  CnKKflnCOK  DHUICS*  'DCfDCD' 

depending  upon  the 

in  which  the  rays  of  light, 
are  equally  refracted  in  all  parts 
of  Ac  •rriifa».  bat  the  refraction  of  at  least  two  meridians  is 


in  which  the  various  parts 


is  that  form  in  winch  the  rays 
are  brought  to  a  focus  on 
through  the  otiier  principal  meridian 
a  point  back  of  the  retina. 
Cempemd  kyptropu  astigmatism  is  that  form  in  which  the 


VISION. 

rays  from  both  meridians  are  focussed  back  of  the  retina,  but  at 
two  different  points. 

(e)  Simple  myopic  astigmatism  is  that  form  in  which  one  merid- 
ian refracts  rays  to  a  point  in  front  of  the  retina,  and  the  other 
principal  meridian  fooisses  rays  upon  the  retina. 

(/)  Compound  hyperopic  astigmatism  is  that  form  in  which  rays 
passing  through  both  principal  meridians  are  brought  to  a  focus  at 
different  points  in  front  of  the  retina. 

(g)  Mixed  astigmatism  is  that  form  in  which  one  meridian  fo- 


FIG.  4$.— Described  in  text 

cusses  rays  in  front  of  the  retina,  and  the  other  focusses  them  back 
of  the  retina. 

In  simple  hyperopic  astigmatism  the  rays  are  focussed  at  2  and 
3  (see  Fig.  45). 

In  compound  hyperopic  astigmatism  the  rays  are  focussed  at  i 
and  2. 

In  simple  myopic  astigmatism  the  rays  are  focussed  at  3  and  4. 

In  compound  myopic  astigmatism  the  rays  are  focussed  at  4 
and  5. 

In  mixed  astigmatism  the  rays  are  focussed  at  2  and  4. 

(/;)  Presbyopia  is  loss  of  accommodative  power  due  to  sclerosing 
of  the  crystalline  lens.  Although  the  process  commences  during 
the  first  year  of  life,  the  lens  does  not  lose  enough  of  its  elasticity 
to  interfere  with  near  vision  until  about  the  age  of  fort}'.  It  is  cor- 
rected by  placing  before  the  eye  a  convex  lens.  Why  ?  Make  a 
diagram. 

CORRECTION  OF  REFRACTIVE  DEFECTS. 

The  Numbering  of  Lenses. — Lenses  are  measured  according  to 
their  refractive  power.  A  lens  whose  focal  distance  is  one  metre 


LABORATORY  MANUAL  OF  PHYSIOLOGY. 

is  taken  as  the  unit  of  measure.  It  is  numbered  i,  and  is  called 
one  diopter  (D).  The  refractive  power  of  a  lens  is  the  inverse  of 
its  focal  distance.  Hence  a  lens  of  2  diopters  has  a  focal  distance 
of  0.50.  What  is  the  focal  distance  of  a  lens  of  4  D?  Given  a 
lens  whose  focal  distance  is  2  metres,  what  is  its  number  ? 

Parallel  rays  of  light,  passing  through  a  convex  lens,  are  made 
to  converge.  Parallel  rays  of  light,  passing  through  a  concave 
lens,  are  made  to  diverge. 

A  cylindric  lens  is  a  lens,  one  or  both  surfaces  of  which  are  seg- 
ments of  a  cylinder.  Rays  of  light,  passing  through  it  in  a  plane 
parallel  to  its  axis,  are  not  bent.  Rays  passing  in  a  plane  perpen- 
dicular to  its  axis,  converge  or  diverge,  according  as  to  whether  the 
cylinder  is  concave  or  convex. 

Lenses  are  designated  plus  (+)  if  they  are  convex,  and  minus 
( — )  if  concave. 

What  forms  of  ametropia  would  cylinders  correct  ? 

OPHTHALMOSCOPY. 

The  ophthalmoscope  is,  in  its  simplest  form,  a  mirror  with  a  hole 
in  it.  The  first  instrument  of  Helmholtz,  in  fact,  consisted  of 
three  thin  plates  of  glass,  fastened  together  and  mounted  in  a  frame, 
at  an  angle  of  56°.  The  whole  object  of  the  instrument  is  to  il- 
luminate the  ocular  fundus  by  reflected  rays  of  light  and  permit 
the  observer  to  inspect  the  illuminated  area.  All  patterns  are 
useful.  The  patterns  of  Loring  and  Morton  are  most  popular. 

Use. — Not  all  the  light  entering  the  pupils  is  absorbed  by  the 
pigmentary  layer  of  the  choroid.  A  certain  amount  returns  from 
the  eye.  If,  therefore,  the  observer's  eye  is  placed  in  the  same 
position  as  the  source  of  illumination,  or  directly  behind  it,  the 
interior  of  the  eye  becomes  visible.  This  is  the  principle  of  the 
ophthalmoscope.  The  mirror,  which  gathers  rays  of  light  from  a 
luminous  point,  becomes  a  secondary  source  of  light  which  is  pro- 
jected into  the  pupil. 

Methods.—  There  are  two,  direct  and  indirect.  In  the  direct, 
the  examiner  places  his  eye  close  to  that  of  the  patient  and  looks 

[192] 


VISION. 


FIG.  46.  —  Use  of  Ophthalmoscope.  Direct 
method.  S,  eye  of  subject ;  £,  eye  of  observer; 
M,  mirror ;  L,  source  of  light. 


directly  upon  the  enlarged  and  upright  details  of  the  fundus.  In 
the  indirect,  the  subject  is  at  an  arm's  length,  and  a  convex  lens  is 
placed  between  the  sub- 
ject's eye  and  the  exam- 
iner's  mirror.  The  image, 
as  obtained,  is  inverted 
and  aerial.  The  two 
methods  differ,  practi- 
cally, in  that  the  direct 
image  is  larger  and  erect, 
in  a  small  field,  while  the 
indirect  image  is  smaller 
and  inverted,  but  in  a 
larger  field.  The  two 
methods  are  explained  in 
the  accompanying  figures 
(46  and  47). 

Examine  the  fundus,  both  by  the  direct  and  indirect  method, 
and  make  an  outline  drawing  showing  the  disc  and  retinal  vessels 
in  each  case. 

PERIMETRY. 

In  contradistinction  to  visual  acuity  which  is  limited  to  the 
macula,  the  function  of  sight  performed  by  the  other  parts  of  the 
retina  is  called  indirect  vision. 

The  limits  of  the  field  of  vision  are  best  obtained  by  an  instru- 
ment, the  perimeter,  but  a  fairly  accurate  map  of  a  field,  not  larger 
than  45°,  may  be  obtained  on  a  blackboard. 

Exercise. — In  the  centre  of  the  blackboard,  in  the  line  of  direct 
vision,  locate  a  dot,  the  point  of  fixation.  Draw  from  the  dot,  as 
a  centre,  a  series  of  circles  whose  distance  from  each  other  shall 
represent  an  angular  distance  of  10°. 

Now  draw  the  meridians  which  will  divide  each  quadrant  into 
at  least  three  subdivisions. 

A  wooden  guide,  twenty  centimetres  long,  should  be  provided 


LABORATORY  MANUAL  OF  PHYSIOLOGY. 

to  regulate  the  distance  of  the  subject's  eyes  from  the  point  of  fixa- 
tion. The  subject,  with  one  eye  screened,  places  himself  directly 
in  front  of  the  point  of  fixation  and  twenty  centimetres  from  it. 
Make  a  test  object  out  of  a  piece  of  white  paper,  one  centimetre 


FIG.  47.— Use  of  Ophthalmoscope.    Indirect  method.    S,  eye  of  subject;  £,  eye  of 
observer;  M,  mirror;  L,  source  of  light;  C,  convex  lens;  /,  image  of  fundus. 

square,  and  affix  it  to  a  black  handle.  Let  the  operator  move  the 
test  object  along  one  meridian,  say  the  horizontal,  from  the  pe- 
riphery toward  the  point  of  fixation.  As  soon  as  the  subject  sees 
the  test  object,  make  a  chalk  mark  on  the  meridian,  denoting  the 
place  where  it  is  first  seen.  In  like  manner  go  over  at  least  eight 
of  the  meridians.  Join  the  points  so  obtained  with  a  line,  and  the 
result  is  the  approximate  field  for  white. 

In  the  same  manner  map  out  the  field  for  blue  and  red. 

Which  field  is  the  largest,  that  for  white,  for  blue,  or  for  red? 
Which  is  the  smallest? 

DRUGS  ACTING  LOCALLY  ON  THE  EYE. 

Those  acting  directly  upon  the  eye  are  divided  into  (i)  mydri- 
atics  (dilators  of  the  pupil),  such  as  atropine,  homatropine,  cocaine, 
scopolamine,  etc. 

(2)  Myotics  (pupil  contractors),  such  as  eserine  and  pilo- 
carpine. 

[194] 


VISION. 

(3)  Cycloplegics  (paralysants  of  the  ciliary  muscles),  such  as 
atropine,  homatropine,  scopolamine,  etc. 

(4)  Anaesthetics  (local),  such  as  cocaine,  eucaine,  holocaine. 
Of  the  mydriatics,  the  first  four  are  in  most  common  use.     They 

differ  from  one  another  in  intensity  and  duration  of  effect,  in  the 
order  named.  The  mydriatics,  as  a  rule,  increase  the  intraocular 
tension. 

Exercise. — Instil  a  drop  of  homatropine  into  a  subject's  eye. 
What  effect  does  it  have  on  the  pupil  ?  Upon  accommodation  ? 
Explain  the  action. 

Instil  a  drop  of  eserine  in  the  same  eye.  What  effect  is  pro- 
duced ? 

Instil  a  drop  of  cocaine  into  another  subject's  eye.  Brush  the 
cornea,  lightly,  with  a  wisp  of  cotton.  Note  the  anaesthesia  and 
the  effect  on  the  pupil.  Which  produces  the  more  complete 
mydriasis,  cocaine  or  homatropine? 


[195] 


INDEX. 


Aberration,  chromatic,  186 

spherical,  186 
Absorption,  121 
of  fat,  137 

Acceleration,  heart-beat,  87 
Accommodation,  visual,  184 
Action  current  of  muscle,  35,  36 

detection     of,     with     tel- 
ephone, 38 
of  frog's  heart,  137 
reflex,  47 
Adaptive  sera,  78 
Adrenalin,  action  on  blood-vessels, 

H7>  J45 

on  heart-beat,  92 
on  muscle  twitch,  45 
on  shock,  117 
on  urine  flow,  169 
^Esthesiometer,  173 
After -load  of  muscle,  26 
Albumin,   intravenous  injection   of, 

169 

Albumins,  differentiation  of,  132 
Albuminuria,  169 
Algae,  i 

Ametropia,  190 
Amoeba,  5 

influence  of  temperature  on,  6 
ingestion  of  foreign  bodies  by,  6 
locomotion  of,  6 
structure  of,  5,  6 
Ampere,  14 

Amyl  nitrite,  effect  on  blood  pressure 
and  kidney  volume,  169 


Amylolytic  action  of  pancreatic  juice, 

136 

Anelectrotonus,  40 
Anode,  14 
Anodic  closing  contraction,  43 

opening  contraction,  43 
Apex  beat  of  heart,  109 
Apparatus,  arrangement  of,  for  clcc- 

trotonus,  39,  40 
for  study  of  ciliary  mo- 
tion, 9 
to  show  action  of  heart 

valves,  10 1 
electrical,  13 
Artificial  respiration,  162 
Ascending  currents,  40 
Asphyxia,  158 
Astigmatism,  190 
Atropine,  action  on  heart,  88 

on  secretion  of  saliva,  124 
Auricles,  record  of  pulsation,  84 
Auscultation,  149 

Battery  cells,  arrangement  of,  15 
Bayliss  and  Starling  on  pancreatic 

secretion,  139 

Bellows  recorder,  Brodie,  167 
Bile  acids,  test  for,  137 

action  of,  137 

pigments,  test  for,  137 
Binocular  fixation,  visual,  185 
Biological  introduction,  i 
Biuret  test,  131 
Bladder,  movements  of,  166 


INDEX. 


Blind  spot,  eye,  187 
Blood,  circulation  of,  80 
coagulation  of,  56 

action  of  peptone  on,  170 
action  of  reagents  on,  57 
calcium  salts  in,  57,  58 
fibrin  in,  56,  58 
fibrinogen  in,  58 
corpuscles  of,  59 
defibrination  of,  58 
fresh,  examination  of,  63 
haemoglobin  of,  73 
laking  of,  58 

microscopic  examination  of,  63 
plasma  of,  56 
pressure,  105 

and  kidney  volume,  168 
effect  of  depressor-nerve 

stimulation  on,  115 
effect  of  hemorrhage  on 

116, 

effect  of  shock  on,  117 
effect     of     vagus  -  nerve 

stimulation  on,  114 
estimation  of  human,  118 
record  of  in  rabbit,  113 
serum,  57 
smears,  64 

specific  gravity  of,  71 
Brain,  frog's,  47 
pigeon's,  52 
motor  areas  of,  53 
Breathing,  bronchial,  149 

vesicular,  149 

Brodie,  bellows  recorder,  167 
Bulbus  arteriosus,  85 

Calcium  salts,  action  on  heart  mus- 
cle, 94 
in    coagulation    of    blood, 

57,  58 
Canals,  semicircular,  54 


Capillary  electrometer,  35 

Carbon  dioxide,  influence  on  plant 

cells,  2,  3 

influence  on  animal  cells,  8 
monoxide,    haemoglobin,    spec- 
trum of,  76 

Cardiac  nerves,  extrinsic,  87,  98 
dissection  of,  87,  98 
stimulation  of,  88,  100 
Cardiogram,  109 
Cardiograph,  84,  109 
Cardio-pneumatic  movements,  152 
Caruncula,  178 
Cells,  blood,  59 

difference  between  animal  and 

plant,  5 
nerve,    discharge    of    impulses 

from,  55 
Centres,  reflex,  49 

vasomotor,  112 
Cerebellum,  removal  of,  52 
Cerebrum,  motor  areas  of,  53 

removal  of,  effect  on   reflexes, 

48 

in  frog,  51 
in  pigeon,  52 

Cervical  sympathetic,  section  of,  115 
Chemical    stimuli    of    muscle    and 

nerve,  22 

Chest  measurements,  151 
Chloral    hydrate,    influence    on    re- 
flexes, 49 

Chloroform,  action  on  heart,  91,  94 
Chlorophyll,  i 

Chorda-tympani  nerve,  123,  124 
Chromatophores,  i,  3 
Cilia,  work  done  by,  10,  n 
Ciliary  arteries,  178 

motion,  effect  of  CO2  on,  n 
effect  of  other  gases  on,  12 
effect  of  temperature  on,  10 
rate  of,  9 

98] 


INDEX. 


Ciliated  epithelium,  g,  10,  n,  12 
Circulation  of  blood,  80 

artificial  schema  of,  102 
in  mesentery  of  frog,  81 
in  respiration,  165 
in  web  of  frog,  80 
mechanics  of,  102 
Coagulation  of  blood,  56 
Cocaine,  action  on  heart,  89 
Commutator,  Pohl's,  39,  40 
Compensatory  pause  of  heart,  97 
Complementary  air,  152 
Conjugation  in  plants,  3 
Conjunctiva,  178 

Contractions,  muscle,  Galvani's  ex- 
periments, 34 

paradoxical,  37 

secondary,  34 

summation  of,  29 
Convergence,  visual,  186 
Cord,  spinal,  diffusion  of  impulses 
in,  48 

hemisection  of,  52 

reflex  centres,  48 
Corpuscles,  blood,  56 

action  of  reagents  on,  62 

classification  of,  66 

differential  count  of,  64 

enumeration  of,  59 

frog's    and    mammalian,    com- 
pared, 64 

iodine  reaction  in,  65 

staining  of,  64 
Curare,  action  of,  25 
Current,  ascending,  40 

constant,  as  a  stimulus,  38,  41 

demarcation,  34 

descending,  40 

effect  on  nerve  irritability,  38 

galvanic,  13 

of  action,  35,  36,  38 

of  injury,  34,  36 


Current,  Pfliiger's  laws,  41,  42 
Currents,  electrical,  regulation  of,  15 

induced,  16 
Cytolysis,  77 

Dare,  haemoglobino meter  of,  68 
Defibrination  of  blood,  58 
Degeneration,  reaction  of,  44 
Deglutition,  mechanism  of,  127 
Demarcation  current,  34 
Depressor  nerve,  100 

effect  of  stimulation  of,  on 

kidney  volume,  168 
Descending  current,  40 
Dextrin,  126 
Dextrose,  125 

intravenous  injection  of,  169 
Diabetes,  pancreatic,  141 
Dialysis,  136 

Diaphragm,  innervation  of,  156 
Digestion,  121 

gastric,  130 

intestinal,  134 

salivary,  125 

Digitalin,  action  on  heart,  90 
Diopter,  192 
Dioptric  system,  181 
Dissection,  frog's  leg,  18 

heart,  82,  97 
Diuresis,  168,  169 

Drum,  arrangement  of,  for  superim- 
posed twitches,  27 
Drugs,  action  on  heart,  88 

action  on  muscle  twitch,  45 
Duct,  lachrymal,  178 

sub  maxillary,  123 

Elasticity  of  muscle,  21 
Electrical  stimuli,  23 
Electrolytes,  14 
Electrometer,  capillary,  35 
Electromotive  force,  14 


199 


INDEX. 


Electrotonus,  38 

Emmetropia,  190 

Emulsification,  134 

Epithelium,  ciliated,  9 

Ergography,  31 

Erythrocytes,  enumeration  of,  59 

Ether,  action  on  heart,  91,  93 

Euglena  viridis,  6 

Excretion,  166 

Extrinsic  cardiac  nerves,  87,  88,  98, 

100 

Eye,  adaptation  for  direction,  184 
for  distance,  184 

appendages  of,  178 

as  an  optical  instrument,  182 

cardinal  points  of,  183 

coats  of,  179 

dissection  of,  178 

drugs  acting  locally  on,  194 

humors  of,  179 

lens  of,  179 

posterior  chamber  of,  179 

suspensory  ligament  of,  179 
Eyeball,  178 


Fatigue,  effect  on  muscle  twitch,  26 
on  tetanus,  36 

of  human  skeletal  muscle,  31 

record  of  curve,  27,  28 
Fats,  absorption  of,  137 

digestion  of,  134 
Fibrin  ferment,  58 

formation  of,  57 
Fibrinogen,  58 
Films,  blood,  staining  of,  64 
Flagellae,  6 

Fleischl,  v.,  hae  mo  meter,  69 
Flow  of  current,  14 

of  liquids,  104 
Foci,  conjugate,  181 
Fovea  centralis,  179 
Fraunhofer  lines,  75 


Fremitus,  vocal,  150 
Fungi,  4 

Galvani,  experiment  with  metals,  34 

without  metals,  13,  34 
Galvanic  cell,  13 

current,  13 
Gastric  digestion,  130 

juice,  132 
Gastrocnemius-sciatic     preparation, 

18 

Glands,  lachrymal,  178 
Meibomian,  178 
salivary,  changes    in,  following 
chorda  stimulation,  124 
dissection  of,  122 
sebaceous,  178 

Glycogen,  preparation  of,  140 
Glycosuria,  169 
Gmelin  test  for  bile  pigments,  137 


Haematin,  73 

spectrum  of,  77 
Haematoporphyrin,  74 

spectrum  of,  77 
Haemin,  73 
Haemocytometer,   Thoma-Zeiss,   59, 

60 

Haemoglobin,  crystals  of,  73 
derivatives  of,  73,  74,  75 

spectra  of,  75 
estimation  of,  68 
reduced,  76 
Haemoglobinometer,  Dare's,  68 

Talqvist's,  68 
Haemolysis,  77 

Haemometer,  v.  FleischFs,  69 
Hammerschlag,    estimation  of   spe- 
cific gravity  of  the  blood,  71 
table    of    comparison    between 
specific    gravity  and    haemo- 
globin, 71 

[200] 


INDEX. 


Heart,  action  current  of,  37 
action  of  frog's,  82,  83 

of  rabbit's,  98 
dissection  of  frog's,  82 

of  rabbit's,  97 
excision  of,  83 
extrinsic  nerves  of,  86,  87 
sounds,  109 
valves,  action  of,  101 
Heart-beat,  action  of  drugs  on,  88 
direct  observation  of,  82,  98 
graphic  record  of,  84 
influence  of  temperature  on,  83, 

85,  118 

nerve  action  on,  87,  100 
reflex  inhibition  of,  88 
Stannius'  experiment,  95 
Heart  muscle,  action  of  salts  on,  94 
compensatory  pause,  97 
maximal  response  of,  96 
refractory  period,  97 
Helmholtz,  ophthalmoscope  of,  192 
Hemisection  of  spinal  cord,  52 
Hemorrhage,  effect  on  blood  press- 
ure, 116 

Holmgren,  test  for  color  vision,  189 
Hooke's  law,  21 
Hyperopia,  190 
Hypoglossal  nerve,  122 

Images,  retinal,  183 

Sanson's,  187 

Impressions,  visual,  duration  of,  188 
Impulse,  nerve,  velocity  of,  24 
Induced  currents,  study  of,  16,  17 
Inductorium,  16 
Infusoria,  6 

Inhibition  of  heart-beat,  87,  88 
Internal  secretion,  140 
Interrupters,  18 
Intestines,  digestion  in,  134 
Involuntary  muscle,  45 


Iodine,  test  for  dextrin,  126 

for  glycogen,  140 

for  starch,  125 
lodophilia,  65 
Irritability  of  nerve  and  muscle,  22 

and  effect  of  constant  cur- 
rent on,  38 
Isometric  contraction  of  muscle,  32 

spring,  graduation  of,  33 
Isotonic  contraction  of  muscle,  33 

Judgments,  visual,  187 

Kanthack  and  Hardy,  classification 

of  leucocytes,  66 
Kathelectrotonus,  40 
Kathode,  14 
Kathodic  opening  contraction,  43 

closing  contraction,  43 
Kidney  volume,  167 

volume  and  blood  pressure,  168 
volume  and  depressor  stimula- 
tion, 1 68 

volume,  effect  of  vagus  stimula- 
tion on,  1 68 

Kronecker,  apparatus  for  ciliary  mo- 
tion, 9 

interrupter,  18 
perfusion  of  frog's  heart,  93 
water  pen,  108 

Lachrymal  ducts,  178 

glands,  178 
Laking  of  blood,  58 
Latent  period  of  stimulation,  123 
Lenses,  179 

numbering  of,  191 
Leucocytes,  classification  of,  66 

differential  count  of,  68 

enumeration  of,  61 

migration  of,  81 

of  frog's  blood,  6 


[201] 


INDEX. 


Leucocytes,  of  human  blood,  64 

staining  of,  64 

varieties  of,  67 
Light,  influence  on  plant  cells,  2,  4 

refraction,  179 
Lipolytic  action  of  pancreatic  juice, 

136 

Liquids,  swallowing  of,  130 
Liver,  formation  of  dextrose  in,  141 

glycogen  of,  140 
Load,  muscle,  25 
LugoPs  solution,  140 

Macrospores,  i 
Manometer,  mercury,  113 
Maximum  response  of  heart  mus- 
cle, 96 

Mechanism  of  circulation,  102 
Medulla,  destruction  of,  in  frog,  48 
Meibomian  glands,  178 
Metabolism,  carbohydrate,  after  pan- 
creas excision,  141 
effect  of  sunlight  on   plant,  3, 

4,5 

MethEemoglobin,  spectrum  of,  76 
Microspores,  i,  2 
Migration  of  leucocytes,  81 
Milk,  digestion  of,  134 
Millon,  reagent  of,  131 
Monocular  fixation,  184 
Mosso,  ergograph  of,  32 

plethysmograph  of,  108 
Motor  areas,  stimulation  of,  53 
Muscarin,  action  of,  on  heart,  90 
Muscle,  action  current  of,  36 

action  of  curare  on,  25 

actual  shortening  of,  31 

demarcation  current  of,  36 

elasticity  of,  20 

electric  phenomena  of,  34 

fatigue  of  frog's,  26 

fatigue  of  human,  31 


Muscle,  Hooke's  law,  21 
involuntary,  45 

irritability  of,  to  stimuli,  22,  23 
isometric  contraction  of,  33 
isotonic  contraction  of,  33 
reaction  of  degeneration  in,  44 
secondary  contraction  of,  34 
single  twitch,   action  of  drugs 

on,  45 
form  of,  23 

influence  of  fatigue  on,  26 
influence  of  load  on,  25 
influence     of    temperature 

on,  26 
summation  of  contractions,  29 

of  stimuli,  28 
tensile  strength  of,  21 
tension,  influence   on    contrac- 
tion, 32 
tetanus  of,  29 
tone,  37 

volume  of  contracting,  28 
work  doneduring  contraction,  31 
Muscle-nerve  physiology,  13 
Muscles,  dissection  of  frog's  leg,  18, 

iQ 
Myopia,  190 


Nerve  cell,  number  of  impulses  dis- 
charged in  given  time,  31,  55 
conductivity,  influence  of  con- 
stant current  on,  38,  40 
electric  phenomena  of,  34 
impulse,  velocity  of,  24 

diffusion  of,  in  cord,  48 
irritability,  to  stimuli,  22,  23 
influence  of  constant  cur- 
rent on,  38,  40 
stimulation  of  human,  42,  44 
Nerves,  cardiac,  frog,  87 

rabbit,  100 
chorda  tympani,  122,  123 

[202] 


INDEX. 


Nerves,  hypoglossal,  122 

laryngeal,  inferior,  128,  129 
superior,  128 

lingual,  122 

optic,  179 

vagus,  87,  100,  114,  128,  129 
Nervous  system,  physiology  of,  47 
Newton's  rings,  61 
Nicotine,  action  on  heart,  90 

on  nerve  cells,  124 
Nitric-acid  test  for  proteids,  131 

Ohm,  15 

Ohm's  law,  14 

Oncometer,  167 

Ophthalmoscopy,  192 

Optical  defects,  186 

Optic  lobes,  removal  of,  in  frog,  48 

nerve,  179 

Optics,  physiology  of,  179 
Oxy-haemoglobin,  spectrum  of,  75 

Pancreas,  excision  of,  141 
Pancreatic  diabetes,  141 

juice,  135 

action  on  fats,  136 
action  on  proteids,  135 
action  on  starches,  136 

secretion,  mechanism  of,  138 
Palpation,  150 
Paramcecium,  7 
Pasteur  fluid  for  yeast,  4 
Pepsin,  action  of,  133 
Peptone,  effect  on  blood  coagulation, 

170 

Peptones,  132 
Peptonuria,  170 
Percussion,  150 
Perfusion  of  frog's  heart,  92 

of  rabbit's  heart,  117 
Perimetry,  193 
Peristalsis,  cesophageal,  127 


Pettenkofer  test  for  bile  acids,  137 
Pfliiger,  laws  of  constant  currents, 

41,  42 

Phrenic  nerve,  156 
Physiological  salt  solution,  diuretic 

action  of,  169 
Pilocarpine,  action  on  heart,  89 

action  on  salivary  secretion,  124 
Pithing,  frog,  10 
Plasma,  blood,  56 

salted,  58 

Plethysrriograph,  108 
Plica  semilunaris,  178 
Porter,  ergograph  of,  32 

plethysmograph  of,  108 
Potassium    salts,    action    on    heart 

muscle,  95 
Presbyopia,  191 
Pressure,  pulmonary,  153 

intrathoracic,  153 
Proteids,  tests  for,  130 

differentiation  of,  132 
Proteoses,  132 
Protococcus,  i,  2 
Protozoa,  5,  6,  7 

effect  of  gases  on,  8,  9 
Pseudopodia  of  amoeba,  6 
Pulse,  record  of,  105,  106 

volume,  107 
Puncta  lachrymalia,  178 

Quotient,  respiratory,  164 

Rabbit,  blood  pressure  of,  27 
Reaction,  iodine,  for  blood,  65 
of  degeneration,  44 
time,  for  sound,  50 
for  touch,  51 
for  vision,  50 
xantho-proteic,  131 
Reflex  action,  in  frog,  47,  48 

centres,  49 
203] 


INDEX. 


Reflex  inhibition  of  heart,  88 

responses,  purposive   character 

of,  49 
time,  48 
Reflexes,  action  of  drugs  on,  49 

diffusion  of  impulses  within  the 

cord,  48 
influence  of  chloral  hydrate  on, 

49 

influence  of  strychnine  on,  49 

inhibitory,  88 

vasomotor,  112 
Refraction,  index  of,  179 
Refractive  defects,  correction  of,  191 
Refractory  period  of  heart  muscle,  97 
Rennin,  action  of,  134 
Reproduction,  3 
Resistance  in  circulation  of  blood,  106 

to  electric  currents,  14 
Respiration,  148 

artificial,  162 

diaphragm  in,  157 

estimation  of  CO2and  H2O  in, 

163 

vagus  nerve  in,  154 
Respiratory  capacity,  151 
centre,  159,  160 
movements,  148,  154 

effect  of  anaemia  on,  159 
effect  of    temperature  on, 

158 

quotient,  164 
sounds,  149 
Retina,  179 

formation  of  images  on,  183 
Rheocord,  15 
Rheostat,  15 

Ring  test  for  proteids,  132 
Riva-Rocci  sphygmomanometer,  119 

Saccharomyces  cerevisiffi,  4 
Saliva,  action  on  starch,  125 


Saliva,  chemical  constituents  of,  125 

secretion  of,  121 
Saponification,  134 
Sartorius-muscle  preparation,  20 
Schema  of  circulation,  102 
Sebaceous  glands,  178 
Secretin,  action  of,  in  pancreatic  se- 
cretion, 139 
Secretion,  gastric,  132 
pancreatic,  138 
salivary,  121 

collection  of,  123 
digestion  by,  125 
effect  of  atropine  on,  124 
effect  of  nicotine  on,  123 
effect  of  pilocarpine  on,  124 
nervous  mechanism  of,  121 
Secretions,  internal,  140 
Semicircular  canals,  54 
Semimembranosus-muscle   prepara- 
tion, 20 
Sensation,  171 

cutaneous,  173 
tactile,  173 

tests  of,  173,  174,  175 
threshold  value  of,  173 
Sense  of  sight,  178 

of  temperature,  176 
Serum,  adaptation  of,  78 

globulicidal  action  of,  77 
Shadows,  inversion  of,  188 
Shock,  action  of  adrenalin  in,  117 

blood  pressure  in,  117 
Sight,  sense  of,  178 
Smear,   blood,   method  of  prepara- 
tion, 64 

Snellen,  test  letters  of,  188 
Sodium  salts,  action  on  heart  mus- 
cle, 93 

Specific  gravity  of  blood,  71 
Spectrum,  absorption,  75 

blood  pigments,  75 
[204] 


INDEX. 


Spectrum,  CO2  haemoglobin,  76 

Fraunhofer  lines  of,  75 

haematin,  77 

haematoporphyrin,  77 

haemoglobin,  reduced,  76 

methsemoglobin,  76 

oxy-haemoglobin,  75 

sodium,  75 

solar,  75 

Sphygmomanometer,  119 
Spinal  cord  as  a  reflex  centre,  48 
hemisection  of,  52 
diffusion  of  impulses  in,  48 
Spirogyra,  2,  3 

Spirometer,  calibration  of,  151 
Stannius'  experiment,  95 
Starch,  digestion  of,  125 

formation  of,  in  plant  cell,  3,  4 
Stimulation,  latent  period  of,  23 
Stimuli,  chemical,  electrical,  mechan- 
ical, 22,  23 

summation  of,  28 

Stimulus  and  sensation,  relation  be- 
tween, 172 

Stomach,  vagus  nerve  in,  129 
Strychnine,  influence  on  reflexes,  49 
Sugars,  test  for,  125 
Summation  of  contractions,  29 

of  stimuli,  28 
Sunlight,  effect  on  organisms,  2 

effect  on  plant  metabolism,  4,  5 
Supplemental  air,  152 
Suprarenal  glands,  144 

extract  of,  145 

effect  on  blood  pressure,  145 
effect  on  heart-beat,  92 
effect  on  muscle  twitch,  45 

removal  of,  in  rabbit,  144 
Suspension    method     of    recording 

heart-beat,  84 
Swallowing,  mechanism  of,  127 

relation  of  vagus  nerve  to,  128 


Sylvester,  method  of  artificial  respira- 
tion, 162 

Sympathetic  nerve,  cervical,  section 
of,  115 

Tactile  sense,  173 
Talqvist,  hoemoglobinometer  of,  68 
Tarsal  cartilages,  eye,  178 
Telephone,  detection  of  action  cur- 
rent with,  23 

Temperature,  influence  of,  on  heart- 
beat, 83,  85,  118 
on  muscle  twitch,  26 
on  plant  growth,  4 
on  tetanus,  30 
sense,  176 

Tenon,  capsule  of,  178 
Tensile  strength  of  muscle,  21 
Tension,    influence    of,   on    muscle 

twitch,  33 

Tetanus,  effect  of  fatigue  on,  30 
effect  of  temperature  on,  30 
genesis  of,  29 
incomplete,  30 
Thermal  stimuli  of  muscle  and  nerve, 

23 

Thigh,  frog's,  dissection  of,  18 
Thoma-Zeiss  haemocytometer,  59 
Thrombin,  58 
Thyroid  feeding,  142 

removal,  142 
Tidal  air,  152 
Tone,  muscle,  artificial,  37 

natural,  38 
Torula,  4 
Touch-spots,  174 
Trypanosoma,  6,  7 
Tuning-fork  interrupter,  18 
Twitch,  muscle,  effect  of  fatigue  on, 
26 

effect  of  temperature  on,  26 

form  of,  23 


INDEX. 


Urea,  diuretic  action  of,  169 
Ureter,  movements  of,  166 
Urine  flow,  167 

Vagus  nerve  in  relation    to    blood 

pressure,  114 
in  relation  to   heart-beat,  87, 

IOC 

in  relation  to  respiration,  154, 

161 

in  relation  to  stomach  move- 
ments, 129 
in     relation     to     swallowing 

movements,  128 
section  of,  161 
Valves,  heart,  action  of,  25 
Vasomotor  centres  in  cord,  112 
in  medulla,  no,  in 
outside  of  cord,  112 
mechanism,  no 
nerves,  115 
reflexes,  112 

Velocity  of  nerve  impulse,  24 
Venae  vorticosae,  178 
Ventricle,  heart,  record  of  contrac- 
tion, 84 
Veratrine,  action  on  muscle  twitch, 

45 
Vision,  178 

abnormal,  190 


Vision,  color  sense,  189 

distant,  188 

light  sense,  189 

miscellaneous  experiments,  187 

near,  189 

normal,  188 

stereoscopic,  185 
Visual  judgments,  187 
Vital  capacity,  152 
Vocal  bands,  movement  of,  130 
Volt,  15 
Volta,  13 

Volume  of  contracting  muscle,  2:, 
Vorticella,  7 

Water,  distilled,  effect  on  plant  life, 

3 

Weber's  law,  173 
Wright  stain  for  blood  corpuscles,  64 

Xantho-proteic  reaction,  131 

Yeast  plant,  4 

growth  of,  4 
influence  of  light  on,  5 
products  of  growth,  5 

Zoospores,  i,  2 
Zygospores,  3 


[206] 


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